CN108937848A

CN108937848A - System for carrying out leukaemia diagnosis and treatment

Info

Publication number: CN108937848A
Application number: CN201710366648.4A
Authority: CN
Inventors: 张超; 井多辉
Original assignee: Hangzhou Hema Biological Technology Co Ltd
Current assignee: Hemachao Biotechnology Shanghai Co ltd
Priority date: 2017-05-23
Filing date: 2017-05-23
Publication date: 2018-12-07
Anticipated expiration: 2037-05-23
Also published as: CN116162704A; CN115927622A; CN115927619A; CN108937848B; CN115927620A; CN115927621A

Abstract

This application involves the system for carrying out leukaemia diagnosis and treatment, which includes: presence and/or the detection module of ratio for being used to detect leukaemia cell for constructing the modeling module of the specific humanized disease model of leukaemic and at least one.The system of the application has the characteristics that specificity is high, can be used for carrying out patient personalized diagnosis and treatment.

Description

System for carrying out leukaemia diagnosis and treatment

Technical field

This application involves a kind of disease treatment systems.System especially for carrying out leukaemia diagnosis and treatment.The system can Predict that drug to the therapeutic effect of patient, and passes through personalized inspection according to performance of the drug in individual specificity's disease model It surveys to track the effect for the treatment of.

Background technique

Leukaemia is a kind of hemopoietic system malignant proliferative disorders, is often referred to largely be proliferated because of leukaemia cell, accumulates institute Caused one kind disease.Due to the complexity of leukemia classification and prognosis process, in order to obtain better therapeutic effect, dialogue blood Disease it is accurate diagnosis with treat accordingly it is imperative.

Show that standardized chemotherapy is invalid to a large amount of patients there are many evidence.Almost 100% patient is being diagnosed trouble Standardized chemotherapy will all be received first after having leukaemia.By taking leukemia of children as an example, only the first stage is just for standard chemotherapy regimen It include using 8 kinds of chemotherapeutics, and all there are strict requirements for the administration time of every kind of drug, and total course for the treatment of is up to 1 year.Chemotherapy at Function is the premise for carrying out other successive treatments, such as bone-marrow transplantation.However the standardized chemotherapy regimen is at least about 25% Leukemia children is invalid, and result is the death rate that this crowd is up to 85%.The most important reason of such case is caused, just It is the individual difference of patient.And standardized therapeutic regimens have ignored this individual difference, provide different individual patients identical Therapeutic scheme.Its result is necessarily: the active drug and ineffective agents in combined chemotherapy cannot be distinguished；Because of invalid chemotherapeutics Patient body is caused huge harm；The mechanism for causing drug resistance is not known that；For drug resistant patient, increasing is clinically taken The method of dosage, higher intensity chemotherapy, injury of the potential increase to patient；It can not provide and embody controlling for patient individual difference Treatment scheme.

On the other hand, effect of the detection cancer cell residual (MRD, Minimal Residual Disease) to assessment treatment There is very important effect with prediction cancer return.In the world, usually using a ten thousandth as boundary, more than the cancer cell of a ten thousandth Residual means very big cancer return probability.Cancer cell is detected in advance, it is meant that is accurately held the state of an illness, is carried out more timely Treatment, thus improve patient survival probability.

Currently, the detection remaining main means of cancer cell are to take Flow Cytometry, that is, carry out cellular level Detection.The precision upper limit of cell-based assay is only a ten thousandth, and this method is easy by cell differentiation and Immune Clone Selection Influence, stability is bad.In addition, this method can not fast, stably, efficiently be applied in actual clinical detection, have Significant limitation.

Summary of the invention

This application provides a kind of systems for carrying out leukaemia diagnosis and treatment.System provided herein can quickly, Reliably, and/or steadily reflect the presence and/or ratio of leukaemia cell in leukaemic.In addition, provided herein System can provide reliable reference for subsequent leukemia treating scheme.System provided herein can be used in candidate Drug screening.In addition, the system of the application can be used for carrying out tailored diagnostics and treatment to subject.For example, the application System and method can be detected by the specific humanized disease model of building subject and with property, and be that subject recommends spy Anisotropic drug candidate and therapeutic scheme.

On the one hand, this application provides the system for carrying out leukaemia diagnosis and treatment, the system includes: a) to model mould Block, is used to construct the specific humanized disease model of subject, and the subject is leukaemic；B) at least one is detected Module is used to detect the presence and/or ratio of leukaemia cell in the subject.The modeling module can include: a1) sample Article unit, it includes the leukaemia cell's suspension for being originated from the subject, the density of leukaemia cell is in the suspension 2x10⁷/ ml to 5x10⁷/ml；A2) radiating element, it includes radioactivity processing units, for immune-deficient mice appropriate Carry out radioactivity processing；A3 it) is inoculated with unit, is used for leukaemia cell's inoculation of suspension liquid at through the radiating element In the immune-deficient mice of reason.At least one described detection module can include: b1) amplification unit, it includes being capable of specificity The reagent of target gene is expanded, to obtain target specificity amplified production.

In some embodiments, the system further includes c) drug candidate screening module.The drug candidate screening Module may include the first drug candidate group and the second drug candidate group；Wherein the first drug candidate group includes selected from the group below One or more drugs: vincristine, dexamethasone, leunase, topoisomerase enzyme inhibitor, purine nucleosides spread out Biology, proteasome inhibitor, mTOR inhibitors, Dasatinib, antibody class drug, all-trans retinoic acid, DNA damage induction Agent, folic acid reductase inhibitor, deacetylate inhibitor, polyceptor tyrosine kinase inhibitor, cytotoxic drug and Wei Jia Acyl phenol amine.The second drug candidate group may include one or more drugs selected from the group below: kinases inhibitor, cell wither Die regulator, DNA distintegrant, cell division inhibitor, MDM2 class oncogene inhibitor, antibody class drug and other chemotherapeutics Object additives.

In some embodiments, the modeling module in the system further includes a4) separative unit, the separation list Member includes monocyte separation medium, for being centrifugated the leukaemia cell of the subject.

In some embodiments, the modeling module in the system further includes a5) T cell removal unit, it uses T cell in the sample that removal is originated from non-T cell type leukaemia subject, to obtain leukaemia cell's suspension.

In some embodiments, the monocyte separation medium is Ficoll, and the centrifuge separation is density gradient Centrifuge separation.

It in some embodiments, include RPMI cell culture medium in leukaemia cell's suspension.

In some embodiments, the T cell being removed is CD3 positive T cell.In some embodiments, institute It states T cell removal unit and contains the magnetic bead comprising CD3 antibody.In some embodiments, the T cell removal unit also includes Highfield.

In some embodiments, the leukaemia is acute lymphatic leukemia, and the immunodeficiency type is small Mouse is NSG mouse.In some embodiments, the leukaemia is acute myeloid leukaemia, and the immune-deficient mice For MISTRG mouse.

In some embodiments, the radioactivity processing unit be X-ray emitter, and its generate dose of radiation For 200cGy to 300cGy.In some embodiments, the radioactivity processing unit is X-ray emitter, and it is generated Dose of radiation be 550cGy to 650cGy.

In some embodiments, the radiating element in the system is arranged to before scheduled inoculation time 12-24 hour in the radioactivity processing is carried out to the immune-deficient mice.

In some embodiments, the age of the immune-deficient mice is 5-7 weeks.

In some embodiments, the modeling module in the system further includes a6) infrared radiation and heating Unit, for carrying out infrared light irradiation and heating to the immune-deficient mice.

In some embodiments, the modeling module in the system further includes a7) monitoring unit, comes for monitoring From the leukaemia cell of the subject in the intracorporal growing state of the immune-deficient mice through being inoculated with.In certain implementations In mode, the monitoring unit includes detecting huCD45+, huCD19+, huCD3+, and/or huCD33 in the mouse peripheral blood The reagent and device of the ratio of+cell.

In some embodiments, the modeling module in the system further includes a8) spleen processing unit, the spleen Processing unit includes resolution element, broken component and levitated element, when huCD45+ in the mouse peripheral blood through being inoculated with, It, can be by described in the resolution element separation when ratio of huCD19+, huCD3+, and/or huCD33+ cell is at least 50% The spleen of mouse is crushed the spleen of the separated mouse by the broken component to obtain spleen cell, and passes through institute It states levitated element and the spleen cell is prepared as leukaemia cell's suspension.

In some embodiments, the modeling module in the system further includes a9) liquid nitrogen, it is used for stored frozen institute State leukaemia cell's suspension.

In some embodiments, the drug candidate screening module further includes monitoring unit, for monitoring from described The leukaemia cell of subject is in the intracorporal growing state of the immune-deficient mice through being inoculated with.In certain embodiments In, the monitoring unit is thin including detecting huCD45+, huCD19+, huCD3+, and/or huCD33+ in the mouse peripheral blood The reagent and device of the ratio of born of the same parents, the monitoring unit can be arranged to, and when the ratio reaches 1%-5%, prompt can be to institute State mouse application drug candidate.

In some embodiments, the monitoring unit include detect huCD45+, huCD19+ in the mouse peripheral blood, The reagent and device of the ratio of huCD3+, and/or huCD33+ cell, the monitoring unit can be arranged to, in medicament administration Afterwards, huCD45+, huCD19+, huCD3+, and/or huCD33+ cell is continued to monitor in the mouse peripheral blood cell The variation of proportion, and prompt according to the variation effect of the drug candidate.

In some embodiments, the monitoring unit is arranged to: after applying the drug candidate, monitoring described HuCD45+, huCD19+, huCD3+, and/or huCD33+ cell proportion in the mouse peripheral blood cell are kept below 1% and when maintaining the ratio at least 2 weeks, then it is effective for prompting the drug candidate.

In some embodiments, the first drug candidate group includes one or more drugs selected from the group below: Changchun New alkali, dexamethasone, leunase, topotecan, clofarabine, Carfilzomib, tesirolimus, Dasatinib, Bortezomib, CD19 antibody, all-trans retinoic acid, adriamycin, ninopterin, SAHA, Sutent, cyclophosphamide and dimension formyl Phenol amine.

In some embodiments, the kinases inhibitor in the second drug candidate group includes being selected from the group One or more drugs: AZD8055, MLN0128, GSK690693, MK-2206, SAR245408, rapamycin, MLN0128、selumetinib、AZD6244、PCI-32765、ibrutinib、SGI-1776、dinaciclib、VS-4718、 Sorafenib, sunitinib and AZD1480.

In some embodiments, the apoptosis regulators in the second drug candidate group include being selected from the group One or more drugs: ABT-263 (navitoclax), ABT-199 (venetoclax), LCL161 and birinapant.

In some embodiments, the DNA distintegrant in the second drug candidate group includes selected from the group below one Kind or a variety of drugs: PR-104, cytarabine (CPX-351), daunorubicin (Vyxeos) and temozolomide.

In some embodiments, the cell division inhibitor in the second drug candidate group includes being selected from the group One or more drugs: eribulin, ispinesib and alisertib.

In some embodiments, the MDM2 class oncogene inhibitor in the second drug candidate group includes choosing From one or more drugs of the following group: MK-8242 (SCH 900242) and RG7112.

In some embodiments, the antibody class drug in the second drug candidate group includes selected from the group below one Kind or a variety of drugs: SAR3419.

In some embodiments, other described chemotherapeutics additives in the second drug candidate group include being selected from One or more drugs of the following group: alvespimycin, AT13387, PF-03084014, RO-4929097, MLN4924, PG11047, CX-5461, BMN-673, selinexor, bortezomib, curaxin and CBL0137.

It in some embodiments, include vincristine in the first drug candidate group, and the vincristine is suitable for The specific humanized disease model of the subject is applied to according to following requirement: being suitable for applying every time by intraperitoneal injection application Dosage is 0.5mg/kg, and application is primary weekly and persistently applies 3-6 weeks.

In some embodiments, wherein including ninopterin, and the ninopterin in the first drug candidate group Suitable for being applied to the leukaemia humanization disease model according to following requirement: being suitable for applying every time by intraperitoneal injection application Amount is 3-6mg/kg, and application is primary every two weeks, and is persistently applied 6-10 weeks.

It in some embodiments, include dexamethasone in the first drug candidate group, and the dexamethasone is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 10-20mg/kg, once-a-day administration and persistently application 3-6 weeks.

It in some embodiments, include adriamycin in the first drug candidate group, and the adriamycin is suitable for basis It is following to require to be applied to the leukaemia humanization disease model: to be suitable for through intravenous injection application, each amount of application is 0.5- 3mg/kg, application is primary weekly and persistently applies 3-6 weeks.

It in some embodiments, include leunase, and the left-handed day in the first drug candidate group Winter amidase is suitable for being applied to the leukaemia humanization disease model according to following requirement: it is suitable for through intraperitoneal injection application, Each amount of application is 500-2000KU/kg, once-a-day administration and persistently application 3-6 weeks.

It in some embodiments, include topotecan in the first drug candidate group, and the topotecan is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 0.1-5mg/kg, once-a-day administration and is persistently applied 2-8 weeks, is discontinued one week after two weeks.

It in some embodiments, include clofarabine in the first drug candidate group, and the clofarabine is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 10-100mg/kg, once-a-day administration and persistently application 3-6 weeks.

It in some embodiments, include Carfilzomib in the first drug candidate group, and the Carfilzomib is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intravenous injection application, each amount of application is 0.1-10mg/kg is applied weekly secondary and is persistently applied 3-6 weeks.

It in some embodiments, include tesirolimus, and the tesirolimus in the first drug candidate group Suitable for being applied to the leukaemia humanization disease model according to following requirement: being suitable for applying every time by intraperitoneal injection application Amount is 5-50mg/kg, once-a-day administration and persistently application 1-3 weeks.

It in some embodiments, include Dasatinib in the first drug candidate group, and the Dasatinib is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for by being administered orally, and each amount of application is 5- 50mg/kg, once-a-day administration and persistently application 2-6 weeks.

It in some embodiments, include bortezomib in the first drug candidate group, and the bortezomib is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 0.1-20mg/kg is applied weekly secondary and is persistently applied 4-8 weeks.

It in some embodiments, include SAR3419 in the first drug candidate group, and the SAR3419 is suitable for root The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 0.5-50mg/kg, application is primary weekly and persistently applies 4-8 weeks.

It in some embodiments, include all-trans retinoic acid, and the total trans dimension in the first drug candidate group Formic acid is suitable for being applied to the leukaemia humanization disease model according to following requirement: being suitable for by intraperitoneal injection application, every time Amount of application is 0.5-10mg/kg, and application is primary weekly and persistently applies 2-6 weeks.

It in some embodiments, include SAHA in the first drug candidate group, and the SAHA is suitable for according to as follows It is required that being applied to the leukaemia humanization disease model: being suitable for through intraperitoneal injection application, each amount of application is 100- 500mg/kg, once-a-day administration and persistently application 3-6 weeks.

It in some embodiments, include Sutent in the first drug candidate group, and the Sutent is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for by being administered orally, and each amount of application is 10- 100mg/kg, once-a-day administration and persistently application 2-6 weeks.

It in some embodiments, include cyclophosphamide in the first drug candidate group, and the cyclophosphamide is suitable for The leukaemia humanization disease model is applied to according to following requirement: being suitable for through intraperitoneal injection application, each amount of application is 30-300mg/kg, once-a-day administration and persistently application 2-6 weeks.

It in some embodiments, include retinamide, and the retinamide in the first drug candidate group Suitable for being applied to the leukaemia humanization disease model according to following requirement: being suitable for applying every time by intraperitoneal injection application Amount is 20-200mg/kg, once-a-day administration and persistently application 2-6 weeks.

In some embodiments, the leukaemia includes that B cell acute lymphoblastic leukemia and/or T cell are acute Lymphocytic leukemia.

In some embodiments, the target gene include one or more genes selected from the group below, its segment or its Variant: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1.

In some embodiments, the target gene includes IgH gene rearrangement, and it is described being capable of specific amplification target The reagent of gene includes combining selected from following one or more groups of primers: 1) forward primer: VH1/7, reverse primer: JH cons； 2) forward primer: VH2, reverse primer: JH cons；3) forward primer: VH3, reverse primer: JH cons；4) forward primer: VH4, reverse primer: JH cons；5) forward primer: VH5, reverse primer: JH cons；6) forward primer: VH6, reverse primer: JH cons；7) forward primer: DH1, DH4, DH5 and DH7, reverse primer: JH cons；8) forward primer: DH2, reverse primer: JH cons；9) forward primer: DH3, reverse primer: JH cons；With 10) forward primer: DH6, reverse primer: JH cons.

In some embodiments, the primer JH cons includes sequence selected from the group below: shown in SEQ ID NO.19 Sequence；With sequence shown in SEQ ID NO.20.In some embodiments, the primer VH1/7 includes sequence selected from the group below Column: shown in SEQ ID NO.1, shown in SEQ ID NO.2 and sequence shown in SEQ ID NO.3.In some embodiments, institute Stating primer VH2 includes sequence selected from the group below: shown in SEQ ID NO.4, shown in SEQ ID NO.5 and shown in SEQ ID NO.6 Sequence.In some embodiments, the primer VH3 includes sequence selected from the group below: shown in SEQ ID NO.7, SEQ ID Shown in NO.8 and sequence shown in SEQ ID NO.9.In some embodiments, the primer VH4 includes sequence selected from the group below Column: shown in SEQ ID NO.10, shown in SEQ ID NO.11 and sequence shown in SEQ ID NO.12.In certain embodiments In, the primer VH5 includes sequence selected from the group below: SEQ ID NO.13 is shown, SEQ ID NO.14 is shown and SEQ ID Sequence shown in NO.15.In some embodiments, the primer VH6 includes sequence selected from the group below: SEQ ID NO.16, Shown in SEQ ID NO.17 and sequence shown in SEQ ID NO.18.In some embodiments, the primer DH1 includes and is selected from The sequence of the following group: shown in SEQ ID NO.21 and sequence shown in SEQ ID NO.22.In some embodiments, the primer DH2 includes sequence selected from the group below: shown in SEQ ID NO.23 and sequence shown in SEQ ID NO.24.In certain embodiments In, the primer DH3 includes sequence selected from the group below: shown in SEQ ID NO.25 and sequence shown in SEQ ID NO.26.? In certain embodiments, the primer DH4 includes sequence selected from the group below: shown in SEQ ID NO.27 and SEQ ID NO.28 institute The sequence shown.In some embodiments, the primer DH5 includes sequence selected from the group below: shown in SEQ ID NO.29 and SEQ Sequence shown in ID NO.30.In some embodiments, the primer DH6 includes sequence selected from the group below: SEQ ID Shown in NO.31 and sequence shown in SEQ ID NO.32.In some embodiments, the primer DH7 includes selected from the group below Sequence: shown in SEQ ID NO.33 and sequence shown in SEQ ID NO.34.

In some embodiments, the target gene includes IgK gene rearrangement, and it is described being capable of specific amplification target The reagent of gene includes combining selected from following one or more groups of primers: 1) forward primer: Vk1, reverse primer: Kdel；2) just To primer: Vk2, reverse primer: Kdel；3) forward primer: Vk3, reverse primer: Kdel；With 4) forward primer: Intron RSS, reverse primer: Kdel.

In some embodiments, the primer Vk1 includes sequence selected from the group below: shown in SEQ ID NO.35 and SEQ Sequence shown in ID NO.36.In some embodiments, the primer Vk2 includes sequence selected from the group below: SEQ ID Shown in NO.37 and sequence shown in SEQ ID NO.38.In some embodiments, the primer Vk3 includes selected from the group below Sequence: shown in SEQ ID NO.39 and sequence shown in SEQ ID NO.40.In some embodiments, the primer Vk4 packet Containing sequence selected from the group below: shown in SEQ ID NO.41 and sequence shown in SEQ ID NO.42.In some embodiments, it states Primer I ntron RSS includes sequence selected from the group below: shown in SEQ ID NO.43 and sequence shown in SEQ ID NO.44.? In certain embodiments, the primer Kdel includes sequence selected from the group below: shown in SEQ ID NO.45 and SEQ ID NO.46 Shown in sequence.

In some embodiments, the leukaemia includes B cell acute lymphoblastic leukemia, the target gene packet Include the gene rearrangement in T cell receptor region；The gene in the T cell receptor region includes one or more selected from the group below: T is thin Born of the same parents' receptor A, T cell receptor D, T cell receptor B and T cell receptor G；And the reagent for capableing of specific amplification target gene Including being combined selected from following one or more groups of primers: 1) forward primer: Vd2, reverse primer: Dd3；2) forward primer: Dd2, Reverse primer: Dd3；3) forward primer: Vd2, reverse primer: Ja29；4) forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；5) forward primer: Vg1 reversely draws Object: Jg1 and Jg2；6) forward primer: Vg2, reverse primer: Jg1 and Jg2；7) forward primer: Vg4, reverse primer: Jg1 and Jg2；8) the more primers of TCRB combine A；9) the more primers of TCRB combine B；C is combined with the more primers of 10) TCRB.

In some embodiments, the primer Vg1 includes sequence selected from the group below: shown in SEQ ID NO.47 and SEQ Sequence shown in ID NO.48.In some embodiments, the primer Vg2 includes sequence selected from the group below: SEQ ID Shown in NO.49 and sequence shown in SEQ ID NO.50.In some embodiments, the primer Vg4 includes selected from the group below Sequence: shown in SEQ ID NO.53 and sequence shown in SEQ ID NO.54.In some embodiments, the primer Jg1 packet Containing sequence selected from the group below: shown in SEQ ID NO.55 and sequence shown in SEQ ID NO.56.In some embodiments, institute Stating primer Jg2 includes sequence selected from the group below: shown in SEQ ID NO.57 and sequence shown in SEQ ID NO.58.In certain realities It applies in mode, the primer Vd2 includes sequence selected from the group below: shown in SEQ ID NO.61 and sequence shown in SEQ ID NO.62 Column.In some embodiments, the primer Dd2 includes sequence selected from the group below: shown in SEQ ID NO.65 and SEQ ID Sequence shown in NO.66.In some embodiments, the primer Dd3 includes sequence selected from the group below: SEQ ID NO.67 institute Show and sequence shown in SEQ ID NO.68.In some embodiments, the primer Ja9 includes sequence selected from the group below: SEQ Sequence shown in ID NO.71.In some embodiments, the primer Ja29 includes sequence selected from the group below: SEQ ID Sequence shown in NO.72.In some embodiments, the primer Ja30 includes sequence selected from the group below: SEQ ID NO.73 Shown in sequence.In some embodiments, the primer Ja48 includes sequence selected from the group below: shown in SEQ ID NO.74 Sequence.In some embodiments, the primer Ja49 includes sequence selected from the group below: sequence shown in SEQ ID NO.75. In some embodiments, the primer Ja52 includes sequence selected from the group below: sequence shown in SEQ ID NO.76.Certain In embodiment, the primer Ja54 includes sequence selected from the group below: sequence shown in SEQ ID NO.77.In certain embodiment party In formula, the primer Ja55 includes sequence selected from the group below: sequence shown in SEQ ID NO.78.In some embodiments, The primer Ja56 includes sequence selected from the group below: sequence shown in SEQ ID NO.79.In some embodiments, described to draw Object Ja57 includes sequence selected from the group below: sequence shown in SEQ ID NO.80.In some embodiments, the primer Ja58 Include sequence selected from the group below: sequence shown in SEQ ID NO.81.In some embodiments, the primer Ja59 includes choosing From the sequence of the following group: sequence shown in SEQ ID NO.82.In some embodiments, the primer Ja61 includes and is selected from the group Sequence: sequence shown in SEQ ID NO.83.

In some embodiments, more primer combination A include one or more sequences selected from the group below: SEQ ID Shown in NO.86, shown in SEQ ID NO.87, shown in sequence, SEQ ID NO.89 shown in SEQ ID NO.88, SEQ ID Shown in NO.90, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 is shown, SEQ ID NO.94 institute Show, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ ID NO.98 is shown, SEQ ID Shown in NO.99, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ ID NO.102 is shown, SEQ ID NO.103 Shown in shown, SEQ ID NO.104, shown in SEQ ID NO.105, shown in SEQ ID NO.106, shown in SEQ ID NO.107, Shown in SEQ ID NO.108, SEQ ID NO.111 is shown, SEQ ID NO.112 is shown, SEQ ID NO.113 is shown, SEQ Shown in ID NO.114, SEQ ID NO.115 is shown, SEQ ID NO.116 is shown, SEQ ID NO.118, SEQ ID NO.122 Sequence shown in shown and SEQ ID NO.123.

In some embodiments, more primer combination B include one or more sequences selected from the group below: SEQ ID Shown in NO.86, SEQ ID NO.87 is shown, SEQ ID NO.88 is shown, SEQ ID NO.89 is shown, SEQ ID NO.90 institute Show, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 is shown, SEQ ID NO.94 is shown, SEQ ID Shown in NO.95, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ ID NO.98 is shown, SEQ ID NO.99 institute Show, shown in SEQ ID NO.100, shown in SEQ ID NO.101, shown in SEQ ID NO.102, shown in SEQ ID NO.103, Shown in SEQ ID NO.104, SEQ ID NO.105 is shown, SEQ ID NO.106 is shown, SEQ ID NO.107 is shown, SEQ Shown in ID NO.108, SEQ ID NO.117 is shown, SEQ ID NO.119 is shown, SEQ ID NO.120 is shown and SEQ ID Sequence shown in NO.121.

In some embodiments, more primer combination C include one or more sequences selected from the group below: SEQ ID Shown in NO.109, SEQ ID NO.110 is shown, SEQ ID NO.111 is shown, SEQ ID NO.112 is shown, SEQ ID Shown in NO.113, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ ID NO.116 is shown, SEQ ID Shown in NO.117, SEQ ID NO.118 is shown, SEQ ID NO.119 is shown, SEQ ID NO.120 is shown, SEQ ID Shown in NO.121, shown in SEQ ID NO.122 and sequence shown in SEQ ID NO.123.

In some embodiments, the leukaemia includes T cell acute lymphoblastic leukemia, the target gene packet Include the gene rearrangement in T cell receptor region；The gene in the T cell receptor region includes one or more selected from the group below: T is thin Born of the same parents' receptor A, T cell receptor D, T cell receptor B and T cell receptor G；And the reagent for capableing of specific amplification target gene Including being combined selected from following one or more groups of primers: 1) forward primer: Vd1, reverse primer: Jd1；2) forward primer: Vd2, Reverse primer: Jd1；3) forward primer: Vd3, reverse primer: Jd1；4) forward primer: Vd2, reverse primer: Dd3；5) forward direction is drawn Object: Dd2, reverse primer: Dd3；6) forward primer: Dd2, reverse primer: Jd1；7) forward primer: Vd2, reverse primer: Ja29； 8) forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；9) forward primer: Vg1, reverse primer: Jg1 and Jg2；10) forward primer: Vg2, reverse primer: Jg1 And Jg2；11) forward primer: Vg3, reverse primer: Jg1 and Jg2；12) forward primer: Vg4, reverse primer: Jg1 and Jg2；13) Forward primer: Sildb, reverse primer: Taldb1；14) the more primers of TCRB combine A；15) the more primers of TCRB combine B；With 16) The more primers of TCRB combine C.

In some embodiments, the primer Vg3 includes sequence selected from the group below: shown in SEQ ID NO.51 and SEQ Sequence shown in ID NO.52.In some embodiments, the primer Vd3 includes sequence selected from the group below: SEQ ID Shown in NO.63 and sequence shown in SEQ ID NO.64.In some embodiments, the primer Vg1 includes selected from the group below Sequence: shown in SEQ ID NO.47 and sequence shown in SEQ ID NO.48.In some embodiments, the primer Vg2 packet Containing sequence selected from the group below: shown in SEQ ID NO.49 and sequence shown in SEQ ID NO.50.In some embodiments, institute Stating primer Vg4 includes sequence selected from the group below: shown in SEQ ID NO.53 and sequence shown in SEQ ID NO.54.In certain realities It applies in mode, the primer Jg1 includes sequence selected from the group below: shown in SEQ ID NO.55 and sequence shown in SEQ ID NO.56 Column.In some embodiments, the primer Jg2 includes sequence selected from the group below: shown in SEQ ID NO.57 and SEQ ID Sequence shown in NO.58.In some embodiments, the primer Vd2 includes sequence selected from the group below: SEQ ID NO.61 institute Show and sequence shown in SEQ ID NO.62.In some embodiments, the primer Dd2 includes sequence selected from the group below: SEQ Shown in ID NO.65 and sequence shown in SEQ ID NO.66.In some embodiments, the primer Dd3 includes and is selected from the group Sequence: shown in SEQ ID NO.67 and sequence shown in SEQ ID NO.68.In some embodiments, the primer Ja9 Include sequence selected from the group below: sequence shown in SEQ ID NO.71.In some embodiments, the primer Ja29 includes choosing From the sequence of the following group: sequence shown in SEQ ID NO.72.In some embodiments, the primer Ja30 includes and is selected from the group Sequence: sequence shown in SEQ ID NO.73.In some embodiments, the primer Ja48 includes sequence selected from the group below Column: sequence shown in SEQ ID NO.74.In some embodiments, the primer Ja49 includes sequence selected from the group below: SEQ Sequence shown in ID NO.75.In some embodiments, the primer Ja52 includes sequence selected from the group below: SEQ ID Sequence shown in NO.76.In some embodiments, the primer Ja54 includes sequence selected from the group below: SEQ ID NO.77 Shown in sequence.In some embodiments, the primer Ja55 includes sequence selected from the group below: shown in SEQ ID NO.78 Sequence.In some embodiments, the primer Ja56 includes sequence selected from the group below: sequence shown in SEQ ID NO.79. In some embodiments, the primer Ja57 includes sequence selected from the group below: sequence shown in SEQ ID NO.80.Certain In embodiment, the primer Ja58 includes sequence selected from the group below: sequence shown in SEQ ID NO.81.In certain embodiment party In formula, the primer Ja59 includes sequence selected from the group below: sequence shown in SEQ ID NO.82.In some embodiments, The primer Ja61 includes sequence selected from the group below: sequence shown in SEQ ID NO.83.

In some embodiments, more primer combination A include one or more sequences selected from the group below: SEQ ID Shown in NO.86, SEQ ID NO.87 is shown, SEQ ID NO.88 is shown, SEQ ID NO.89 is shown, SEQ ID NO.90 institute Show, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 is shown, SEQ ID NO.94 is shown, SEQ ID Shown in NO.95, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ ID NO.98 is shown, SEQ ID NO.99 institute Show, shown in SEQ ID NO.100, shown in SEQ ID NO.101, shown in SEQ ID NO.102, shown in SEQ ID NO.103, Shown in SEQ ID NO.104, SEQ ID NO.105 is shown, SEQ ID NO.106 is shown, SEQ ID NO.107 is shown, SEQ Shown in ID NO.108, SEQ ID NO.111 is shown, SEQ ID NO.112 is shown, SEQ ID NO.113 is shown, SEQ ID Shown in NO.114, SEQ ID NO.115 is shown, SEQ ID NO.116 is shown, SEQ ID NO.118 is shown, SEQ ID Shown in NO.122 and sequence shown in SEQ ID NO.123.

In some embodiments, the primer Vd1 includes sequence selected from the group below: shown in SEQ ID NO.59 and SEQ Sequence shown in ID NO.60.In some embodiments, the primer Jd1 includes sequence selected from the group below: SEQ ID Shown in NO.69 and sequence shown in SEQ ID NO.70.In some embodiments, the primer Sildb includes and is selected from the group Sequence: sequence shown in SEQ ID NO.84.In some embodiments, the primer Taldb1 includes sequence selected from the group below Column: sequence shown in SEQ ID NO.85.

In some embodiments, the leukaemia is B cell acute lymphoblastic leukemia.In certain embodiments In, the leukaemia is T cell acute lymphoblastic leukemia.

In some embodiments, the detection module in the system further includes identification unit, the identification unit packet Containing the reagent and device for identifying the target specificity amplified production, to identify the target specificity amplified production In be originated from the subject leukaemia cell characteristic amplified production.

In some embodiments, the identification unit includes to carry out gel electrophoresis to the target specificity amplified production Reagent and device needed for separation.

In some embodiments, the detection module in the system further includes analytical unit, the analytical unit packet Containing the reagent and device for analyzing the characteristic amplified production, to obtain the leukaemia cell for being specific to the subject Characteristic gene sequence.

In some embodiments, the analytical unit includes to carry out nucleic acid sequencing and sequence to the characteristic amplified production Reagent and device needed for column analysis.

In some embodiments, the analytical unit includes protecting in the nucleic acid sequence for identify the characteristic amplified production Reagent and device needed for keeping property nucleic acid sequence and subject's specific nucleic acid sequence, to obtain, to be specific to the subject white The characteristic gene sequence of blood disease cell.

In some embodiments, the detection module in the system further includes recognition unit, the recognition unit packet Reagent and device containing the characteristic gene sequence for capableing of specific recognition and/or the amplification subject, to judge The presence and/or ratio of residual leukemic cell in the subject.

In some embodiments, the recognition unit includes that specific amplification is specific to the subject leukaemia cell Characteristic gene sequence reagent or device.

In some embodiments, the recognition unit also includes the probe and energy in conjunction with non-characteristic gene sequence Enough expand the reagent or device of non-characteristic gene sequence.

In some embodiments, the non-characteristic gene sequence includes being originated from one of the following group or several genes Nucleic acid sequence: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1.

In some embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from IgH, and it is described can The reagent or device for expanding non-characteristic gene sequence include sequence selected from the group below: shown in SEQ ID NO.124, SEQ ID Shown in NO.125, SEQ ID NO.126 is shown, SEQ ID NO.127 is shown, SEQ ID NO.128 is shown, SEQ ID Shown in NO.129, shown in SEQ ID NO.130 and sequence shown in SEQ ID NO.131.In some embodiments, described non- Characteristic gene sequence includes the nucleic acid sequence from IgH, and the probe in conjunction with non-characteristic gene sequence includes choosing From the sequence of the following group: shown in SEQ ID NO.173, SEQ ID NO.174 is shown, SEQ ID NO.175 is shown, SEQ ID Shown in NO.176, SEQ ID NO.177 is shown, SEQ ID NO.178 is shown, SEQ ID NO.179 is shown and SEQ ID Sequence shown in NO.180.In some embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from IgK, And the reagent that can expand non-characteristic gene sequence or device include sequence selected from the group below: SEQ ID NO.132 institute Show, shown in SEQ ID NO.133, shown in SEQ ID NO.134, shown in SEQ ID NO.135, shown in SEQ ID NO.136, Shown in SEQ ID NO.137, SEQ ID NO.138 is shown, SEQ ID NO.139 is shown, SEQ ID NO.140 is shown and SEQ Sequence shown in ID NO.141.In some embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from IgK Column, and the probe with non-characteristic gene sequence ining conjunction with includes sequence selected from the group below: SEQ ID NO.181 is shown, SEQ Shown in ID NO.182, SEQ ID NO.183 is shown, SEQ ID NO.184 is shown, SEQ ID NO.185 is shown, SEQ ID Shown in NO.186, SEQ ID NO.187 is shown, SEQ ID NO.188 is shown, SEQ ID NO.189 is shown and SEQ ID Sequence shown in NO.190.In some embodiments, the non-characteristic gene sequence includes the core from T cell receptor G Acid sequence, and the reagent that can expand non-characteristic gene sequence or device include sequence selected from the group below: SEQ ID Shown in NO.142, SEQ ID NO.143 is shown, SEQ ID NO.144 is shown, SEQ ID NO.145 is shown, SEQ ID Shown in NO.146, SEQ ID NO.147 is shown, SEQ ID NO.148 is shown, SEQ ID NO.149 is shown, SEQ ID Shown in NO.150 and sequence shown in SEQ ID NO.151.In some embodiments, the non-characteristic gene sequence includes Nucleic acid sequence from T cell receptor G, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below Column: SEQ ID NO.191 is shown, SEQ ID NO.192 is shown, SEQ ID NO.193 is shown, SEQ ID NO.194 is shown, Shown in SEQ ID NO.195, SEQ ID NO.196 is shown, SEQ ID NO.197 is shown, SEQ ID NO.198 is shown, SEQ Shown in ID NO.199 and sequence shown in SEQ ID NO.200.In some embodiments, the non-characteristic gene sequence Nucleic acid sequence including being originated from T cell receptor D and/or T cell receptor A, and described can expand non-characteristic gene sequence Reagent or device include sequence selected from the group below: SEQ ID NO.152 is shown, SEQ ID NO.153 is shown, SEQ ID Shown in NO.154, shown in SEQ ID NO.155, shown in SEQ ID NO.156 and sequence shown in SEQ ID NO.157.At certain In a little embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from T cell receptor D and/or T cell receptor A, And the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: shown in SEQ ID NO.201, SEQ ID Shown in NO.202, SEQ ID NO.203 is shown, SEQ ID NO.204 is shown, SEQ ID NO.205 is shown and SEQ ID Sequence shown in NO.206.In some embodiments, the non-characteristic gene sequence includes the core from T cell receptor B Acid sequence, and the reagent that can expand non-characteristic gene sequence or device include sequence selected from the group below: SEQ ID Shown in NO.158, SEQ ID NO.159 is shown, SEQ ID NO.160 is shown, SEQ ID NO.161 is shown, SEQ ID Shown in NO.162, SEQ ID NO.163 is shown, SEQ ID NO.164 is shown, SEQ ID NO.165 is shown, SEQ ID Shown in NO.166, SEQ ID NO.167 is shown, SEQ ID NO.168 is shown, SEQ ID NO.169 is shown and SEQ ID Sequence shown in NO.170.In some embodiments, the non-characteristic gene sequence includes the core from T cell receptor B Acid sequence, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: SEQ ID NO.207 institute Show, shown in SEQ ID NO.208, shown in SEQ ID NO.209, shown in SEQ ID NO.210, shown in SEQ ID NO.211, Shown in SEQ ID NO.212, SEQ ID NO.213 is shown, SEQ ID NO.214 is shown, SEQ ID NO.215 is shown, SEQ Shown in ID NO.216, shown in SEQ ID NO.217, shown in SEQ ID NO.218 and sequence shown in SEQ ID NO.219.? In certain embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from Tal 1, and described can expand non-spy The reagent or device of sign property gene order include sequence selected from the group below: shown in SEQ ID NO.171 and SEQ ID NO.172 institute The sequence shown.In some embodiments, the non-characteristic gene sequence includes the nucleic acid sequence from Tal 1, and described Probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: shown in SEQ ID NO.220 and SEQ ID Sequence shown in NO.221.

In some embodiments, the detection system includes 2 or 2 or more detection modules, wherein at least one Detection module is used to before the subject receives treatment detect the presence and/or ratio of wherein leukaemia cell, and wherein At least one detection module is used to detect presence and/or the ratio of wherein leukaemia cell after the subject receives the treatment Example.

In some embodiments, the detection module includes treatment prompt unit, according in subject detected The presence of leukaemia cell and/or ratio prompt the successive treatment scheme used to corresponding subject.

In some embodiments, when the ratio of leukaemia cell in subject detected is 1/10⁴It is described when following Treatment prompt unit prompt is described to treat successfully and without carrying out additional treatment.In some embodiments, when it is detected by The ratio of leukaemia cell is 1/10 in examination person⁴When above, the treatment prompt unit prompt treatment is not perfect and needs Carry out other treatments or raising treatment intensity.In some embodiments, when the ratio of leukaemia cell in subject detected Example is 1/104 or more and 1/10³When following, the treatment prompt unit prompt can carry out bone-marrow transplantation hand to shown subject Art.In some embodiments, when the ratio of leukaemia cell in subject detected is 1/10³When above, the treatment Prompt unit prompt is unsuitable for carrying out bone marrow transplant to the subject, and should continue to carry out other control to the subject Treat or improve treatment intensity.

In some embodiments, other treatments are mentioned selected from the monitoring unit of the drug candidate screening module It is shown as the effective drug candidate, or the therapy similar with its function and/or effect and/or drug.

Those skilled in the art can from detailed description below in easily have insight into the other aspects of the disclosure and excellent Gesture.The illustrative embodiments of the disclosure only have been shown and described in detailed description below.As those skilled in the art will recognize Know, content of this disclosure enables those skilled in the art to be modified without de- disclosed specific embodiment Spirit and scope from invention involved by the application.Correspondingly, the description in the drawing and description of the application is only example Property, rather than be restrictive.

Detailed description of the invention

The specific features invented involved in the application are as shown in the appended claims.By reference in more detail below The illustrative embodiments and attached drawing of description better understood when the application involved the characteristics of inventing and advantage.To attached drawing letter Want specification as follows:

The flow cytometry analysis schematic diagram that blood sample to Leukemia Patients is analyzed is shown in Fig. 1.

The schematic diagram that flow cytometry analysis is carried out to the leukaemia cell for eliminating T cell is shown in Fig. 2.

The schematic diagram that flow cytometry analysis is carried out to the T cell being trapped in screening pipe is shown in Fig. 3.

The grade scale schematic diagram assessed pharmaceutical efficacy is shown in Fig. 4.

The result that gene expression analysis is carried out to the disease model of the application is shown in Fig. 5.

Fig. 6 is the detection remaining schematic diagram of subject leukaemia cell.

Fig. 7 is the result schematic diagram of expression of specific gene in the leukaemia cell for show subject.

Fig. 8 is the result schematic diagram for showing patient-specific DNA sequence dna in detection subject leukaemia cell.

Fig. 9 is the result schematic diagram by qPCR reaction detection subject leukaemia cell.

Figure 10 is the operating process schematic diagram of the leukaemia diagnosis and therapy system of the application.

Figure 11 is the schematic diagram of each functional unit relationship in the leukaemia diagnosis and therapy system of the application.

Specific embodiment

Illustrate the embodiment of the present application by particular specific embodiment below, those skilled in the art can be by this Specification disclosure of that is easily realized by other advantages and effect of the present application.

In this application, term " leukaemia " (Leukemia) is often referred to largely be proliferated because of leukaemia cell, accumulates and drawn The disease risen, usually starts in marrow.The leukaemia may include that B cell acute lymphoblastic leukemia and/or T cell are anxious Property lymphocytic leukemia.

In this application, term " about " typically refers to become in the range of specified numerical value above and below 0.5%-10% It is dynamic, for example, specified numerical value above and below 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, 5%, 5.5%, it changes in the range of 6%, 6.5%, 7%, 7.5%, 8%, 8.5%, 9%, 9.5% or 10%.

Modeling module

In this application, term " modeling module " typically refers to the specific human for constructing the subject for suffering from leukaemia The functional unit of source disease model.For example, the modeling module may include comprising the leukaemia cell from the subject The sample unit of suspension, the radiating element comprising radioactivity processing unit and for by leukaemia cell's inoculation of suspension liquid To the inoculation unit of the immune-deficient mice handled through the radiating element.

For example, the modeling module may include obtaining the sample bearing device of Samples subjects (for example, blood taking needle etc. obtains The device of the carrying sample such as device, test tube of sample), processing Samples subjects obtain the leukaemia cell suspension of subject Sample processing device (for example, solution and test tube of suspension leukaemia cell)；Immune-deficient mice appropriate is put The radioactivity processing unit (for example, radiotherapy X-ray accelerator) and immune-deficient mice of penetrating property processing are (for example, NSG Mouse, MISTRG mouse)；Leukaemia cell's suspension can be inoculated into the immune-deficient mice handled through the radiating element In classification inoculation apparatus (for example, syringe etc.).

The modeling module can also include the separative unit that can separate Samples subjects.For example, the modeling module It may include a point cellifugal reagent (for example, monocyte separation medium), point cellifugal device (for example, centrifuge).It is described to build Mould module can also include that removal is originated from the T cell in the sample of non-T cell type leukaemia subject to obtain the white blood The T cell removal unit of sick cell suspending liquid.For example, the modeling module may include highfield (for example, magnetic field strength is not less than About 0.05 tesla), CD3 positive T cell screening reagent (for example, by the coated magnetic bead of CD3 monoclonal antibody).The modeling mould Block can also include the infrared radiation and heating unit being irradiated using infrared light supply to individual, for example, the modeling module It may include infrared light supply (for example, 175 watts of infrared lamps).The modeling module can also include monitoring from the white of the subject Monitoring unit of the blood disease cell in the intracorporal growing state of the immune-deficient mice through being inoculated with.For example, the modeling mould Block may include monitoring huCD45 in the mouse peripheral blood through injection inoculation⁺、huCD19⁺、huCD3⁺, and/or huCD33⁺Carefully The monitoring device (for example, flow cytometer) and monitoring reagent of the ratio of born of the same parents are (for example, anti-CD45 antibody, anti-CD 19 antibodies, anti- CD3 antibody and/or anti-CD 33 antibody).The modeling module can also include being handled the spleen of the disease model Spleen processing unit.For example, the modeling module may include resolution element for separating the spleen of the mouse (for example, hand Art scissors), for being crushed the spleen of the separated mouse with obtain spleen cell broken component (for example, metal screen, Plastic filter screen) and for the spleen cell to be prepared as to leukaemia cell's suspension levitated element (for example, RPMI train Support base).The modeling module can also include liquid nitrogen.

In this application, term " subject " is often referred to the individual with certain disease characterization, and disease characterization can be with The symptom for referring to disease can also refer to the harmful physiological status that cannot be changed in prevention implementations.The individual may include Male and/or female, generally comprise people or non-human animal, such as non-human mammal.In some embodiments, the individual Including but not limited to people, dog, cat, horse, sheep, goat, pig, ox, rabbit, rat, mouse, monkey etc..For example, the subject can be with For human patients.

In this application, term " the specific humanized disease model of subject " is often referred to that subject's specificity can be embodied , by humanization modified disease model.For example, display characterization subject's genius morbi, by the inclusion of and/or expression be originated from Tissue, cell and/or the gene of human body and the disease model being humanized.In some embodiments, the subject is special Property humanization disease model includes subject's specificity leukaemia humanized mouse model.For example, the white blood of subject specificity Sick humanized mouse model includes the leukaemia cell from the subject, and huCD45 in the peripheral blood of the mouse model +, the ratio of huCD19+, huCD3+, and/or huCD33+ cell based on total number of cells, can at least about 5%, at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, at least about 85% or at least about 90%.

For example, the specific humanized disease model of subject (for example, leukaemia humanization disease model) can lead to Cross following method to prepare: it is outstanding that the sample of bone marrow from the subject (for example, leukaemic) a) is prepared as cell Liquid, and the cell suspension is centrifuged using monocyte separation medium；B) it obtains thin comprising leukaemia cell Born of the same parents' layer simultaneously prepares leukaemia cell's suspension；C) judge whether the leukaemia is T cell type leukaemia；It is white for non-T cell type Blood disease, removal obtain leukaemia cell's suspension to be seeded by the T cell in b) leukaemia cell's suspension of acquisition, and Make the whole density 2x10 of leukaemia cell in leukaemia cell's suspension to be seeded⁷/ ml to 5x10⁷/ml；For T cell Type leukaemia prepares leukaemia cell's suspension to be seeded from leukaemia cell's suspension by b) obtaining, wherein leukaemia The whole density of cell is 2x10⁷/ ml to 5x10⁷/ml；D) immune deficiency appropriate is selected according to the type of leukemia of the patient Type mouse, and radioactivity processing is carried out to the immune-deficient mice；It e) will be by the leukaemia to be seeded of c) acquisition Pallium cell injection be inoculated into d) described in through radioactivity handle immune-deficient mice in.

In this application, term " sample unit " is typically referred to comprising the sample from subject, such as the white blood of subject The functional unit of sick cell suspending liquid.The sample unit may include sample bearing device and the processing for obtaining Samples subjects Samples subjects obtain the sample processing device of leukaemia cell's suspension of subject.For example, the sample bearing device can Including obtaining the device of sample (for example, blood taking needle, vacuum blood collection tube, urine collecting, saliva collecting device, collecting dung Device etc.) and carrying sample device (for example, test tube, centrifuge tube etc.).For example, the sample processing device may include that suspension is white The solution of blood disease cell is (for example, contain fetal calf serum solution, the CaCl of about 10%DMSO₂Solution) and test tube.

The sample unit may include leukaemia cell's suspension from the subject, white blood in the suspension The density of sick cell can be 1x10⁷/ ml to 5x10⁷/ ml, 1.5x10⁷/ ml to 5x10⁷/ ml, 2x10⁷/ ml to 5x10⁷/ ml, 2.5x10⁷/ ml to 5x10⁷/ ml, 3x10⁷/ ml to 5x10⁷/ ml, 3.5x10⁷/ ml to 5x10⁷/ ml, 4x10⁷/ ml to 5x10⁷/ ml, 4.5x10⁷/ ml to 5x10⁷/ ml, 4.8x10⁷/ ml to 5x10⁷/ ml, 4.9x10⁷/ ml to 5x10⁷/ ml, 2x10⁷/ ml is extremely 2.5x10⁷/ ml, 2x10⁷/ ml to 3x10⁷/ ml, 2x10⁷/ ml to 3.5x10⁷/ ml etc..

In this application, term " sample " typically refer to directly or indirectly to be derived from subject, can therefrom obtain The sample for the sample unit of leukaemia cell's suspension.In some embodiments, directly it is derived from the institute of subject Sample is stated without being further processed, for example, blood can be obtained from the peripheral circulation system of subject (for example, passing through blood sampling Needle).The sample may include for example, blood, urine, excrement, saliva, cerebrospinal fluid and sweat etc..The sample it is non-limiting Example includes blood (or the blood obtained from any anatomical location (for example, tissue, the circulatory system, marrow) of subject Ingredient-is for example, leucocyte, red blood cell, blood platelet), the cell, skin, the heart that are obtained from any anatomical location of subject Dirty, lung, kidney, expiratory air, marrow, excrement, sperm, vaginal secretion, the tissue fluid from tumor tissues, breast, pancreas, brain ridge Liquid, tissue, brush,throat, biopsy article, placental fluids, amniotic fluid, liver, muscle, smooth muscle, bladder, gall-bladder, colon, intestines, brain, chamber Liquid, phlegm, purulence, micropopulation (micropiota), meconium, milk, prostate, esophagus, thyroid gland, serum, saliva, urine, stomach Liquid and digestive juice, tear, ocular fluids, sweat, mucus, earwax, oil, glandular secretion object, spinal fluid, hair, nail, skin are thin Born of the same parents, blood plasma, nose swab or nasopharynx washing lotion, spinal fluid, Cord blood, lymph and/or other excretas or bodily tissue.Certain In embodiment, the sample is the sample of bone marrow from subject, for example, it includes the bone marrow cell of subject and/or blood Cell.In another example the sample is the sample of bone marrow of leukaemic, wherein leukaemia cell accounts for 90% or more.

In this application, term " suspension " typically refers to be often referred to one or more cells to be scattered in formed in liquid Cell suspending liquid, wherein cell can separate as individual or in by no more than about 50, no more than about 40, not more than About 30, no more than about 20, no more than about 10 or less cells composition agglomerate in.

In this application, term " leukaemia cell " typically refers to the jejune white of candidate stem cell morbid state differentiation generation Cell.For example, the function of leukaemia cell's usually not normal cell, can also there is very strong proliferative capacity.

In this application, term " cell density " or " density " typically refer to contain in unit volume in cell suspending liquid The number of cell.For example, the density of leukaemia cell can be 2x10 in the suspension⁷/ ml to 5x10⁷/ ml is 2.5x10⁷/ ml to 5x10⁷/ ml is 3x10⁷/ ml to 5x10⁷/ ml is 3.5x10⁷/ ml to 5x10⁷/ ml is 4x10⁷/ ml is extremely 5x10⁷/ ml is 4.5x10⁷/ ml to 5x10⁷/ ml is 4.8x10⁷/ ml to 5x10⁷/ ml is 4.9x10⁷/ ml to 5x10⁷/ml。

Cell suspending liquid can be obtained by using culture medium in the application, as long as the culture medium can make the cell Normal growth.In some embodiments, the culture medium also makes cell be in the state to suspend.For example, can be used RPMI cell culture medium prepares leukaemia cell's suspension.In another example DMEM culture medium, IMDM culture medium, HAMF12 can be used Culture medium, MEM culture medium, 199 cell culture mediums, MDSS2 cell culture medium etc..

In this application, term " radiating element " typically refers to the structural unit comprising radioactivity processing unit.For example, institute Stating radiating element can be the functional unit for carrying out radioactivity processing to immune-deficient mice appropriate.

In this application, term " radioactivity processing unit " typically refers to that (such as alpha ray, β is penetrated by that can issue ray Line, gamma-rays, X-ray etc.) the device that is irradiated of radioactive source.For example, radiotherapy X-ray accelerator, radiotherapy With electron-beam accelerator, medical accelerator, X-ray deep therpy apparatus, medical X-ray CT machine, heavy particle therapy accelerator, proton Therapeutic device, irradiation devices accelerator, accelerator for neutron production etc..

In this application, term " immune deficient mice " typically refers to one or more immune system constituents presence The mouse of defect, this defect can be as congenital hereditary be mutated and/or by artificial means caused by.In this application, institute Stating immune deficient mice can be T- lymphocyte function deficient mice, B- lymphocyte function deficient mice, N cell function Deficient mice, combined immunodeficiency mouse etc..For example, the immunodeficient mouse can be NSG mouse, MISTRG mouse, naked Mouse, CBA/N mouse, Beige mouse, Scid mouse etc..The age of the immune-deficient mice can for 5-7 weeks (for example, Age is 5-6 weeks, and the age is 5-6.5 weeks, and the age is 5.5-6.5 weeks), the age of the immune-deficient mice can also be 5- 8 weeks, 5-9 weeks, 5-10 weeks, 4-10 weeks, 4-12 weeks, 3-10 weeks, 3-12 weeks.

In this application, term " NSG mouse " is often referred to the mouse of NOD-scid IL-2R γ knockout, lacks Mature T cell, B cell and natural killer cells.

In this application, term " MISTRG mouse " is often referred to expression granular macrophage colony stimulating factor of human body (M-CSF), people Interleukin-13 (IL-3)/granulocyte-macrophage colony stimutaing factor (GM-CSF), people's signal adjusting protein alpha (SIRPa), people's blood Platelet generates plain (TPO) and erythropoietin (EPO) gene, but does not express recombination- activating genes 2 (RAG2) or interleukin-22 The mouse of receptor y (IL2Rg).

In this application, when the leukaemia is acute lymphatic leukemia, the immune-deficient mice can For NSG mouse.When the leukaemia is acute myeloid leukaemia, the immune-deficient mice can be MISTRG mouse.

Before injecting leukaemia cell's suspension 12-24 hour (such as 10-24 hours, 10-36 hours, 10-72 Hour, 8-24 hours, 8-36 hours, 8-72 hours) it is interior to the immune-deficient mice progress radioactivity processing.

The method of the radioactivity processing can be such as X-ray radiation, gamma Rays.Those skilled in the art can The condition of radioactivity processing is adjusted according to the type of mouse, condition of inoculation etc..For example, X-ray can be used (for example, at least The X-ray of 1MV, at least 2MV, at least 3MV, at least 4MV, at least 5MV or higher energy) mouse is irradiated.It is described The intensity of X-ray can be for example, at least 100cGy/ minutes, at least 150cGy/ minutes, at least 200cGy/ minutes, at least 250cGy/ minutes, at least 300cGy/ minutes, at least 310cGy/ minutes, at least 320cGy/ minutes, at least 325cGy/ minutes, At least 340cGy/ minutes, at least 360cGy/ minutes, at least 380cGy/ minutes, at least 400cGy/ minutes or higher.Carry out institute Used dose of radiation can be about 50cGy to 100cGy, about 100cGy to 150cGy, about 150cGy when stating radioactivity processing To 200cGy, about 200cGy to 250cGy, about 250cGy to 300cGy, about 300cGy to 350cGy, about 350cGy to 400cGy, About 400cGy to 450cGy, about 450cGy are to 500cGy, about 500cGy to 550cGy, about 550cGy to 600cGy, about 600cGy To 650cGy, about 650cGy to 700cGy, about 700cGy to 750cGy, about 750cGy to 800cGy, about 800cGy to 850cGy Or it is higher.For example, can about 6-8 hours before injecting leukaemia cell's suspension, about 8-12 hours, about 12-16 it is small When, about 16-20 hours, about 20-24 hours, about 24-28 hours, immune lacked to described in about 28-32 hours or a longer period of time Swaged mouse carries out the radioactivity processing.

In some embodiments, when the immune-deficient mice is NSG mouse, X-ray can be used to carry out it Radioactivity processing.Dose of radiation can be about 200cGy to about 300cGy, for example, about 250cGy.For example, the X- that 4MV can be used is penetrated Line, and its intensity is about 325cGy/ minutes.

In some embodiments, when the immune-deficient mice is MISTRG mouse, it can be used X-ray to it Carry out radioactivity processing.Dose of radiation can be about 550cGy to about 650cGy, for example, about 600cGy.For example, can be used 4MV's X-ray, and its intensity is about 325cGy/ minutes.

In this application, term " inoculation unit " is typically referred to for being inoculated into leukaemia cell's suspension through the radiation Functional unit in the immune-deficient mice of cell processing.For example, the inoculation unit may include classification inoculation apparatus, as injection is set Standby (for example, syringe, needleless injector etc.), fully automatic inoculating machine etc..

In this application, term " inoculation " typically refers to cell to be seeded, solution or reagent etc. passing through injection device It is input to the injection intracorporal process of object.For example, the cell to be seeded, solution or reagent etc. can be from subject's Leukaemia cell's suspension.

The preparation method of the leukaemia humanization disease model may also include following step: g) when through injection inoculation HuCD45 in the mouse peripheral blood⁺、huCD19⁺、huCD3⁺, and/or huCD33⁺When the ratio of cell is at least 50%, use Separative unit separates the spleen of the mouse, and it is broken to make it, and makes to be suspended in cell culture through the cell of the broken spleen In base, to obtain the cell suspension from the spleen；H) using monocyte separation medium come to the cell from spleen Suspension is centrifuged；I) obtain include leukaemia cell cellular layer, make the cell in the cellular layer be suspended in containing In the fetal calf serum of 10%DMSO, to prepare final cell densities as 2x10⁷/ ml to 5x10⁷The stock solution of/ml；And it j) will be by i) The stock solution stored frozen obtained is spare in liquid nitrogen.

In this application, term " separative unit " is typically referred to using each component in mixture in physical property or chemically Difference in matter makes each component distribute to different area of space or be sequentially allocated in different times by method appropriate To the functional unit in the same space region.For example, the separative unit can separate the sample of the subject.The separation is single Member may include point cellifugal reagent and/or point cellifugal device.For example, described point of cellifugal reagent can be thin for monokaryon Born of the same parents' separating liquid, for example, Ficoll.In another example described point of cellifugal device can be centrifuge, as high speed freezing centrifuge, Room temperature supercentrifuge, table top general purpose centrifuge etc..

In this application, term " monocyte separation medium " typically refers to the reagent for separating blood monocytes. In some embodiments, the monocyte separation medium utilizes the volume and/or the differences such as form and/or density of cell, right Monocyte is separated and/or is purified.For example, the monocyte separation medium is Ficoll.In another example the monocyte Separating liquid can be glucan and aqueous solution, the Percoll solution of Angiografin etc..For example, described is separated into centrifugation Separation.The centrifuge separation can separate for density gradient centrifugation.

In this application, term " T cell removal unit " typically refers to tested from non-T cell type leukaemia for removing T cell in the sample of person, to obtain the functional unit of leukaemia cell's suspension.For example, the T cell removal is single Member may include highfield and/or T cell separation agent.In some embodiments, the T cell being removed is CD3 positive T Cell (T lymphocyte (CD3+)).In order to remove the CD3 positive T cell, the T cell removal unit may also include CD3 sun Property T cell screening reagent.For example, the T cell separation agent may include heparin, lymphocyte separation medium, cell culture fluid etc.. The CD3 positive T cell screening reagent may include the magnetic bead comprising CD3 antibody.For example, the magnetic bead is to be resisted by CD3 monoclonal The coated magnetic bead of body.It for example, the magnetic bead can be the coated magnetic bead of anti-human CD3mAb, or is AntiCD3 McAb/anti- CD28 monoclonal The coated magnetic bead of antibody.In some embodiments, the CD3 positive T cell screening reagent can be the magnetic of Miltenyi company Pearl

In this application, term " highfield " typically refers to be often referred to magnetic field strength not less than about 0.05 tesla, not low In about 0.1 tesla, not less than about 0.3 tesla, not less than about 0.5 tesla, not less than about 0.55 tesla, not less than about 0.6 tesla is not less than about 0.7 tesla, is not less than about 0.8 tesla, is not less than about 0.9 tesla, is special not less than about 1.0 Si La, it is not less than about 2.0 teslas, is not less than about 3.0 teslas or higher magnetic field.

In this application, term " removal T cell " typically refers to after being operated by removal, T cell in monocyte group Ratio no more than about 0.5%, no more than about 0.4%, no more than about 0.3%, no more than about 0.2% or be not greater than about 0.1%.

In this application, term " non-T cell type leukaemia " is typically referred to as acute B-cell type leukaemia, acute marrow are thin The type of leukemia in addition to T cell type leukaemia such as born of the same parents' leukaemia, chronic B cell type leukaemia, chronic myelocytic leukemia.

In this application, term " infrared radiation and heating unit " typically refers to be irradiated individual using infrared light supply Functional unit.For example, the infrared radiation and heating unit may include infrared light supply.Wherein, the infrared light supply refers to wavelength Greater than a certain range of electromagnetic radiation of red wavelengths, such as 0.78~1000 μm.The infrared light supply may include near-infrared, in Infrared or far infrared light source.The wavelength of the near-infrared light source can be 0.78~1.4 μm, 0.78~1.3 μm, 0.78~1.2 μ M, 0.78~1.1 μm, 0.78~1.0 μm, 0.78~0.9 μm, 0.78~0.85 μm, 0.78~0.8 μm.The mid-infrared light source Wavelength can be 1.4~3 μm, 1.5~3 μm, 1.6~3 μm, 1.7~3 μm, 1.8~3 μm, 1.9~3 μm, 2.0~3 μm, 2.2~3 μm, 2.4~3 μm, 2.6~3 μm, 2.8~3 μm, 2.9~3 μm.The wavelength of the far infrared light source can be 3~ 1000 μm, 5~1000 μm, 7~1000 μm, 10~1000 μm, 50~1000 μm, 100~1000 μm, 200~1000 μm, 300 ~1000 μm, 400~1000 μm, 500~1000 μm, 600~1000 μm, 700~1000 μm, 800~1000 μm, 900~ 1000 μm, 950~1000 μm, 980~1000 μm, 990~1000 μm.For example, the infrared light supply is 175 watts of infrared lamps.

In this application, term " monitoring unit " typically refers to exist for monitoring from the leukaemia cell of the subject The functional unit of the intracorporal growing state of the immune-deficient mice through being inoculated with.For example, the monitoring unit may include prison Survey huCD45 in the mouse peripheral blood through injection inoculation⁺、huCD19⁺、huCD3⁺, and/or huCD33⁺The ratio of cell Monitoring device.For example, the detection device can be flow cytometer.In another example the monitoring unit may also include monitoring warp HuCD45 in the mouse peripheral blood of injection inoculation⁺、huCD19⁺、huCD3⁺, and/or huCD33⁺The monitoring of the ratio of cell Reagent.For example, the monitoring reagent can be anti-CD45 antibody, anti-CD 19 antibodies, anti-cd 3 antibodies and/or anti-CD 33 antibody.

In the specific humanized disease model of the subject, the leukaemia cell from the patient can be monitored in institute State the intracorporal growing state of mouse.In some embodiments, by the mouse peripheral blood of the monitoring through injection inoculation huCD45⁺、huCD19⁺、huCD3⁺, and/or huCD33⁺The ratio of cell exists to monitor the leukaemia cell from the patient The intracorporal growing state of mouse, so that the huCD45 in the peripheral blood of the mouse model prepared⁺、huCD19⁺、 huCD3⁺, and/or huCD33⁺The ratio of cell reach the range (for example, at least about the 5% of total number of cells, at least about 10%, At least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, At least about 50%, at least about 55%, at least about 60%, at least about 65%, at least about 70%, at least about 75%, at least about 80%, At least about 85% or at least about 90%).For example, monitoring can be passed through when the leukaemia of the patient is B cell type leukaemia HuCD19 in the mouse peripheral blood through injection inoculation⁺The ratio of cell exists to monitor the leukaemia cell from the patient The intracorporal growing state of mouse.For example, can be passed through by monitoring when the leukaemia of the patient is T cell type leukaemia HuCD3 in the mouse peripheral blood of injection inoculation⁺The ratio of cell monitors the leukaemia cell from the patient in institute State the intracorporal growing state of mouse.For example, can be connect by monitoring through injection when the leukaemia of the patient is myelogenous leukemia HuCD33 in the mouse peripheral blood of kind⁺The ratio of cell monitors the leukaemia cell from the patient in the mouse Intracorporal growing state.

In this application, term " spleen processing unit " typically refer to the spleen of the disease model (such as mouse) into The functional unit of row processing.For example, in the mouse peripheral blood through being inoculated with huCD45+, huCD19+, huCD3+, and/or The ratio of huCD33+ cell be at least 50% (at least about 50%, at least about 55%, at least about 60%, at least about 65%, at least About 70%, at least about 75%, at least about 80%, at least about 85% or at least about 90%) when, can get spleen cell, Jin Ertong It crosses the spleen processing unit and spleen cell is prepared as leukaemia cell's suspension.The spleen processing unit may include Resolution element (for example, surgical scissors, ligature, sterile funnel etc.), broken component are (for example, ultrasonic oscillator, high pressure homogenate Machine, metal screen, plastic filter screen etc.) and levitated element (for example, RPMI culture medium, the fetal calf serum containing about 10%DMSO are molten Liquid, CaCl₂Solution), wherein the resolution element is used to separate the spleen of the mouse；The broken component can be crushed through separating The mouse spleen to obtain spleen cell；It is thin that the spleen cell can be prepared as the leukaemia by the levitated element Born of the same parents' suspension.

In this application, term " liquid nitrogen " typically refers to the nitrogen of liquid.

Drug candidate screening module

In this application, term " drug candidate screening module " is typically referred to for the specific humanized disease of the subject Disease model provides the drug candidate of specificity for subject, to carry out the functional unit of accurate diagnoses and treatment.For example, described Drug candidate screening module may include the first drug candidate group containing one or more drugs, containing one or more drugs Second drug candidate group.For example, the drug candidate screening module may include vincristine, dexamethasone, left-handed asparagine Enzyme, topoisomerase enzyme inhibitor, purine nucleosides analog derivative, proteasome inhibitor, mTOR inhibitors, Dasatinib, antibody Class drug, all-trans retinoic acid, DNA damage inducer, folic acid reductase inhibitor, deacetylate inhibitor, polyceptor junket ammonia Acid kinase inhibitor, cytotoxic drug and retinamide.Such as, the drug candidate screening module may include selected from the group below One or more drugs: vincristine, leunase, topotecan, clofarabine, Carfilzomib, replaces dexamethasone Sirolimus, Dasatinib, bortezomib, CD19 antibody, all-trans retinoic acid, adriamycin, ninopterin, SAHA, Buddhist nun of relaxing replace One of Buddhist nun, cyclophosphamide and retinamide are a variety of.In another example the drug candidate screening module may include that albumen swashs Enzyme inhibitor, apoptosis regulators, DNA distintegrant, cell division inhibitor, MDM2 class oncogene inhibitor, antibody class One of drug and other chemotherapeutics additives are a variety of.

The drug candidate screening module can also include leukaemia cell of the monitoring from the subject through being inoculated with The intracorporal growing state of the immune-deficient mice monitoring unit.Therefore, the drug candidate screening module may include The leukaemia cell from the subject is detected in the inspection of the intracorporal growing state of the immune-deficient mice through being inoculated with It surveys element (for example, flow cytometer), judgement is carried out to the growing state that the detecting element detects and determines the candidate medicine Feedback element (the example of the level results of the decision element (for example, CPU processor) of grade where object and the display decision element Such as, display).

It in this application, may include one or more drug candidates in the drug candidate screening module, for according to need It will be to the specific humanized disease model application of the subject.Each drug candidate for including in the drug candidate screening module Dosage, dosage form and method of application etc., determined according to the characteristics of described drug candidate itself.

In this application, term " drug candidate " is often referred to the substance that some possibility are used as drug, these substances can To be assessed by method appropriate, to obtain the information of its biological activity, pharmacological action and effect etc..

In this application, term " the first drug candidate group " typically refers to the collection being made of one or more drug candidates It closes, can be used for receiving screening in the first stage.The first stage can be the leukemia chemotherapy stage to routinize.Described first Drug candidate group may include the conventional drug for leukemia chemotherapy.For example, the first drug candidate group may include being selected from One or more drugs of the following group: vincristine, dexamethasone, leunase, topoisomerase enzyme inhibitor, purine core Glycoside derivative, proteasome inhibitor, mTOR inhibitors, Dasatinib, antibody class drug, all-trans retinoic acid, DNA damage Inducer, folic acid reductase inhibitor, deacetylate inhibitor, polyceptor tyrosine kinase inhibitor, cytotoxic drug and Retinamide.In some embodiments, the first drug candidate group includes one or more drugs selected from the group below: long Spring new alkali, leunase, topotecan, clofarabine, Carfilzomib, tesirolimus, is replaced up to sand dexamethasone Buddhist nun, bortezomib, CD19 antibody, all-trans retinoic acid, adriamycin, ninopterin, SAHA, Sutent, cyclophosphamide and dimension Formyl phenol amine.

In this application, term " the second drug candidate group " typically refers to the collection being made of one or more drug candidates It closes, can be used for receiving screening in second stage.The second stage can be in the first rank of the leukemia chemotherapy to routinize After section.The second drug candidate group may include one or more drugs selected from the group below: kinases inhibitor, cell wither Die regulator, DNA distintegrant, cell division inhibitor, MDM2 class oncogene inhibitor, antibody class drug and other chemotherapeutics Object additives.

In this application, term " protease inhibitors " typically refer to can with it is some on protease molecule activated centre Group combines, and declines prolease activity, or even disappears, but the substance for being denaturalized protease itself.The protease inhibits Agent may include leupeptin, antipain, chymotrypsin chalone, suppression elastoser aldehyde, suppression pepsin element, phosphamide element.Example Such as, the protease inhibitors can be include one or more drugs selected from the group below: AZD8055, MLN0128, GSK690693、MK-2206、SAR245408、rapamycin、MLN0128、selumetinib、AZD6244、PCI-32765、 Ibrutinib, SGI-1776, dinaciclib, VS-4718, sorafenib, sunitinib and AZD1480.

In this application, term " apoptosis regulators " typically refers to adjust the object of Apoptosis generation and degree Matter.Apoptosis refers generally to the autonomous orderly death of the cell controlled by gene.The apoptosis regulators can pass through adjusting The approach such as the crack conditions of nucleus DNA adjust apoptosis process.For example, the apoptosis regulators may include being selected from down Group one or more drugs: ABT-263 (navitoclax), ABT-199 (venetoclax), LCL161 and birinapant。

In this application, term " DNA distintegrant " typically refers to the reagent for capableing of decomposing D NA.For example, the reagent can lead to It crosses the modes such as DNA crosslinking, the nitrogen-atoms on modifying DNA ring and/or the outer oxygen groups of ring and carrys out decomposing D NA.For example, the DNA is decomposed Agent may include one or more drugs selected from the group below: PR-104, cytarabine (CPX-351), daunorubicin (Vyxeos) and temozolomide.

In this application, term " cell division inhibitor " typically refers to be able to suppress fissional substance.Cell point It splits and refers generally in proliferation process, being divided and increasing its quantity by a cell is the mistake of two or more cells Journey.For example, the cell division agent can inhibit the interaction between FtsZ and ZipA.For example, the cell division inhibitor can Including one or more drugs selected from the group below: eribulin, ispinesib and alisertib.

In this application, term " MDM2 class oncogene inhibitor " typically refers to inhibit the expression of MDM2 class oncogene Substance.The expression product MDM2 albumen of the MDM2 class oncogene can inhibit the normal biology of p53 with p53 protein binding Function leads oncogenic generation.And p53 is a kind of very important human tumor suppressor, it have control DNA repair, The functions such as cell-cycle arrest, Apoptosis.For example, the MDM2 class oncogene inhibitor may include one kind selected from the group below Or a variety of drugs: MK-8242 (SCH 900242) and RG7112.

In this application, term " antibody class drug " typically refers to the antibody class drug for being suitable for treating leukaemia.For example, institute Stating antibody class drug may include one or more drugs selected from the group below: SAR3419.

In this application, term " other chemotherapeutics additives " is typically referred to for assisting during leukemia chemotherapy Enhance the substance of chemotherapeutic efficacy.For example, other described chemotherapeutics additives may include one or more drugs selected from the group below: Alvespimycin, AT13387, PF-03084014, RO-4929097, MLN4924, PG11047, CX-5461, BMN-673, Selinexor, bortezomib, curaxin and CBL0137.

In this application, its dosage and administration time can be adjusted by the characteristic of the drug candidate itself.For example, institute Stating drug candidate may include vincristine, and apply the vincristine to the model as follows: pass through intraperitoneal injection Application, each amount of application are 0.5mg/kg, and application is primary weekly and persistently applies 3-6 weeks.For example, the drug candidate includes ammonia First folic acid, and the ninopterin is applied to the model as follows: it is applied by intraperitoneal injection, each amount of application is 3- 6mg/kg, once-a-day administration are applied every other week and are persistently applied 6-10 weeks.For example, the drug candidate includes dexamethasone, and The dexamethasone to be applied to the model as follows: being applied by intraperitoneal injection, each amount of application is 10-20mg/kg, Once-a-day administration and persistently application 3-6 weeks.For example, the drug candidate includes adriamycin, and as follows to the mould Type applies the adriamycin: being applied by intravenous injection, each amount of application is 0.5-3mg/kg, and application is primary weekly and persistently applies With 3-6 weeks.For example, the drug candidate includes left-handed asparagine, and described left-handed to model application as follows Asparagine: being applied by intraperitoneal injection, and each amount of application is 500-2000KU/kg, once-a-day administration and persistently applies 3-6 Week.For example, the drug candidate includes topotecan, and the topotecan is applied to the model as follows: passing through Intraperitoneal injection application, each amount of application are 0.1-5mg/kg, once-a-day administration and are persistently applied 2-8 weeks, are stopped after two weeks Medicine one week.For example, the drug candidate includes clofarabine, and the clofarabine is applied to the model as follows: It is applied by intraperitoneal injection, each amount of application is 10-100mg/kg, once-a-day administration and persistently application 3-6 weeks.For example, institute Stating drug candidate includes Carfilzomib, and applies the Carfilzomib to the model as follows: being applied by intravenous injection With each amount of application is 0.1-10mg/kg, applies weekly secondary and persistently applies 3-6 weeks.For example, the drug candidate includes Tesirolimus, and the tesirolimus is applied to the model as follows: it is applied by intraperitoneal injection, is applied every time Amount is 5-50mg/kg, once-a-day administration and persistently application 1-3 weeks.For example, the drug candidate includes Dasatinib, and with The Dasatinib as described under type to model application: by being administered orally, each amount of application is 5-50mg/kg, is applied daily With it is primary and persistently apply 2-6 weeks.For example, the drug candidate includes bortezomib, and applied as follows to the model With the bortezomib: being applied by intraperitoneal injection, each amount of application is 0.1-20mg/kg, applies weekly secondary and persistently applies With 4-8 weeks.For example, the drug candidate includes SAR3419, and the SAR3419 is applied to the model as follows: logical Intraperitoneal injection application is crossed, each amount of application is 0.5-50mg/kg, and application is primary weekly and persistently applies 4-8 weeks.For example, described Drug candidate includes all-trans retinoic acid, and applies the all-trans retinoic acid to the model as follows: passing through abdominal cavity Injection application, each amount of application are 0.5-10mg/kg, and application is primary weekly and persistently applies 2-6 weeks.For example, candidate's medicine Object includes SAHA, and applies the SAHA to the model as follows: being applied by intraperitoneal injection, each amount of application is 100-500mg/kg, once-a-day administration and persistently application 3-6 weeks.For example, the drug candidate includes Sutent, and with such as Under type applies the Sutent to the model: by being administered orally, each amount of application is 10-100mg/kg, is applied daily With it is primary and persistently apply 2-6 weeks.For example, the drug candidate includes cyclophosphamide, and applied as follows to the model With the cyclophosphamide: being applied by intraperitoneal injection, each amount of application is 30-300mg/kg, once-a-day administration and is persistently applied With 2-6 weeks.For example, the drug candidate includes retinamide, and the dimension formyl is applied to the model as follows Phenol amine: being applied by intraperitoneal injection, and each amount of application is 20-200mg/kg, once-a-day administration and persistently application 2-6 weeks.

In this application, term " monitoring unit " typically refers to exist for monitoring from the leukaemia cell of the subject The functional unit of the intracorporal growing state of the immune-deficient mice through being inoculated with.For example, the monitoring unit may include inspection The leukaemia cell from the subject is surveyed in the detection of the intracorporal growing state of the immune-deficient mice through being inoculated with Life element (for example, flow cytometer, for example, antibody needed for detection specific cell) and the detecting element is detected Long situation carries out the decision element (for example, CPU processor, operation program) of grade where judgement determines the drug candidate.Institute Stating monitoring unit may also include the feedback element for showing the level results of the decision element (for example, display, display screen, language Sound output device etc.).

The leukaemia cell of the detecting element detection from the subject is small in the immunodeficiency type through being inoculated with The intracorporal growing state of mouse, and the growing state that will test is transferred to the decision element.The decision element is set according to it It is fixed, determine the drug candidate where grade condition, the growing state obtained to transmission judges, obtains grade As a result, simultaneously the level results can be transferred to the feedback element.The feedback element leads to the level results that transmission obtains The usable condition that the forms such as image, sound feed back the drug candidate out to the subject is crossed, to control subsequent clinic It treats and reliable reference is provided.

The monitoring unit can be by detecting the leukaemia cell's in the specific humanized disease model of subject Ratio (for example, passing through the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in monitoring mouse peripheral blood) And/or its variation is to assess the drug effect of the drug candidate.The monitoring unit may include detecting the mouse periphery The reagent and device of the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in blood.For example, the monitoring is single Member may include flow cytometer, detect the ELISA kit etc. of CD45, CD19, CD3 and/or CD33.It is used in the monitoring unit In the reagent and dress that detect the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in the mouse peripheral blood HuCD45+, huCD19+, huCD3+, and/or huCD33+ cell can be continued to monitor in institute after applying drug candidate by setting The variation of proportion in mouse peripheral blood cell is stated, and prompts the effect of the drug candidate according to the variation.

When the monitoring unit detect in the mouse peripheral blood huCD45+, huCD19+, huCD3+, and/or The ratio of huCD33+ cell reach 1%-5% (such as reach 1.2%-5%, 2%-5%, 2.5%-5%, 3%-5%, 3.5%-5%, 4%-5%, 4.5%-5%) when, then it is assumed that drug candidate can be applied to the mouse.In another example the monitoring Unit can be arranged to, and when the ratio reaches 1%-5%, prompt can apply drug candidate to the mouse.When the monitoring Unit detects that the ratio of huCD45+, huCD19+, huCD3+ and/or huCD33+ cell in the mouse peripheral blood keeps low In 1% (such as less than 0.9%, 0.8%, 0.7%, 0.6%, 0.5%, 0.4%, 0.3%, 0.2%, 0.1%, 0.05%), And maintain the ratio at least 2 weeks (for example, at least 3 weeks, at least 4 weeks, at least 5 weeks, at least 6 weeks, at least 7 weeks, at least 8 weeks, at least 9 Week, at least 10 weeks) when, then it is assumed that the drug candidate is effective.In another example the monitoring unit can be arranged to, when the ratio is protected When holding lower than 1% and maintaining the ratio at least 2 weeks, it is effective for prompting the drug candidate.

According to clinical settings, system described herein can also formulate the stringent mark much higher than common statistical standard It is quasi-.During administration, it is thin that human leukemia in the specific humanized disease model of subject is monitored using the monitoring unit Born of the same parents' ratio in peripheral blood in a model, and according to the ratio, drug candidate is divided into 6 grades.In certain embodiment party In formula, the monitoring unit may include feedback element, and the feedback element and detecting element information communication, the feedback element can Grade where showing the drug candidate.For example, 6 grades may respectively be:

1 progressive disease 1 (PD1) of grade: to the specific humanized disease model of subject substantially without therapeutic effect.Example Such as, the monitoring unit detect the ratio of huCD45+ cell in the mouse peripheral blood in 7 days (such as at most 6 days, 5 It, in 4 days, 3 days) reach at least 8% (being for example, at least 8.5%, 9%, 9.5%, 10%) and in 12 days (for example, at most In 11 days, 10 days, 9 days, 8 days) reach at least 25% (being for example, at least 26%, 27%, 28%, 29%, 30%).

2 progressive disease 2 (PD2) of grade: very limited to the therapeutic effect of the specific humanized disease model of subject. For example, the monitoring unit detect the ratio of huCD45+ cell in the mouse peripheral blood in 7 days (such as at most 6 days, 5 It, in 4 days, 3 days) reach at least 6% (being for example, at least 6.5%, 7%, 7.5%, 7.8%) and in 21 days (for example, at most In 20 days, 19 days, 18 days, 17 days) reach at least 20% (being for example, at least 21%, 22%, 23%, 24%, 25%).

3 stability disease (SD) of grade: limited to the therapeutic effect of the specific humanized disease model of subject.For example, The monitoring unit detect the ratio of huCD45+ cell in the mouse peripheral blood in 7 days (such as at most 6 days, 5 days, 4 It, in 3 days) reach at least 3% (being for example, at least 3.5%, 4%, 4.5%, 5%) and in 21 days (such as at most 20 days, 19 It, in 18 days, 17 days) reach at least 10% (being for example, at least 10.5%, 11%, 11.5%, 12%).

Alleviate (PR) in class 4 part: having certain therapeutic effect to the specific humanized disease model of subject, can make The ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell drops to 0% in the mouse peripheral blood, and (for example, 6 days, 5 days, 4 days, 3 days) keep 0% ratio in 1 week.For example, the monitoring unit detects the mouse periphery The ratio of huCD45+ cell reaches in 0%, and 1 week in 7 days (such as at most 6 days, 5 days, 4 days, 3 days) (for example, 6 in blood It, 5 days, 4 days, 3 days) keep 0% ratio, while in 21 days (such as at most 20 days, 19 days, 18 days, 17 days) at most For 3% (such as being at most 2.5%, 2%, 1.5%, 1%).

Class 5 complete incidence graph (CR): there is good therapeutic effect to the specific humanized disease model of subject, can make The ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell drops to 0% in the mouse peripheral blood, and It keeps within 2 weeks or so (for example, 20 days, 19 days, 18 days, 17 days, 16 days, 14 days, 13 days, 12 days, 11 days, 10 days, 9 days, 8 days) 0% ratio.For example, the monitoring unit detects the ratio of huCD45+ cell in the mouse peripheral blood (example in 7 days As in 6 days, 5 days, 4 days, 3 days) reach 0%, and 2 weeks or so (for example, 20 days, 19 days, 18 days, 17 days, 16 days, 14 days, 13 It, 12 days, 11 days, 10 days, 9 days, 8 days) ratio that keeps 0%, while (such as 27 days, 26 days, 25 days, 24 days in 28 days It is interior) it is at most 2% (such as being at most 1.5%, 1%, 0.5%, 0.1%).

Class 6 continued complete remission (MCR): significantly excellent treatment imitates the specific humanized disease model of subject Fruit can be such that the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in the mouse peripheral blood drops to 0%, and 3 weeks or more (for example, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 It, 40 days, 45 days) keep 0% ratio.For example, the monitoring unit detects huCD45+ cell in the mouse peripheral blood Ratio reach 0% in 7 days (such as in 6 days, 5 days, 4 days, 3 days), and 3 weeks or more (for example, 21 days, 22 days, 23 days, 24 days, 25 days, 26 days, 27 days, 28 days, 29 days, 30 days, 35 days, 40 days, 45 days) ratio that keeps 0%, while in 42 days (such as in 41 days, 40 days, 38 days, 36 days) be at most 1% (such as be at most 0.5%, 0.4%, 0.3%, 0.2%, 0.1%).

The division of above-mentioned 6 grades is capable of providing reliable drug test data, and the drug candidate of this specificity is applied Clinical treatment for subject.The candidate that system described herein will be screened according to the drug candidate screening module Drug recommends the application of subject's clinic as the clinical medicine of most suitable subject.

Detection module

In this application, term " detection module " typically refer to detect in the subject presence of leukaemia cell and/ Or the functional unit of ratio.The detection module may include specific amplification target gene to obtain target specificity amplification Product amplification unit (such as, it may include material needed for carrying out the PCR amplification and reagent, as PCR instrument, nucleic acid polymerase, Reverse transcriptase, dNTP, Mg²⁺Buffer needed for ion, amplified reaction etc.).

For example, the detection module, which may also include, can identify in the target specificity amplified production from described The characteristic amplified production of the leukaemia cell of subject identification unit (such as, it may include it is isolated be originated from subject it is white Material and reagent described in the characteristic amplified production of blood disease cell, such as electrophoresis apparatus, electrophoresis tank, gel, electrophoretic buffer). For example, the detection module may also include the characteristic gene sequence that can obtain the leukaemia cell for being specific to the subject The analytical unit of column (for example, may include the reagent and device for analyzing the characteristic amplified production, survey by such as sequencing primer Sequence kit, sequence analysis programs etc.).For example, the detection module, which may also include, can judge to remain in the subject The presence of leukaemia cell and/or the recognition unit of ratio are (for example, may include that specific amplification is specific to the white blood of the subject The reagent or device of the characteristic gene sequence of sick cell, if qPCR reaction needed for material and reagent, as qPCR instrument,Green I, archaeal dna polymerase, dNTP, amplification buffer etc.；Sequence with optical activation when such as hybridizing with amplified production Column specific oligonucleotide probe, such as fluorescence probe).For example, may also include can be according to detected for the detection module The presence of leukaemia cell and/or ratio prompt prompt the treatment for the successive treatment scheme that corresponding subject uses in subject Unit.For example, may include detect subject in leukaemia cell presence and/or ratio detecting element (for example, fluidic cell Instrument), and the decision element judged to the result of presence and/or ratio that the detecting element detects is (for example, at CPU Manage device, operation program).The treatment prompt unit may also include the prompt member for the result of judgement for showing the decision element Part (for example, display, display screen, instantaneous speech power etc.).

In this application, term " amplification unit " typically refers to specific amplification target gene to obtain target specificity The functional unit of amplified production.In this application, the amplification unit may include material and examination needed for carrying out the PCR amplification Agent and/or the report agent (for example, detectable nucleic acid binding reagents) of one or more detection amplified productions.For example, the expansion Increasing unit can be used or including nucleic acid polymerase, reverse transcriptase, dNTP, Mg²⁺Buffer needed for ion, amplified reaction and/or Detect the report agent etc. of amplified production.

In this application, term " amplification " typically refers to amplification target nucleic acid and generates amplified production.The amplification of nucleic acid can be with It is linear, exponential or combinations thereof.Amplification can be emulsion-based or being not based on emulsion.Nucleic acid amplification method Non-limiting example includes reverse transcription, primer extend, polymerase chain reaction, ligase chain reaction, helicase dependent amplification, non- Non-symmetric amplification, rolling circle amplification and multiple displacement amplification (MDA).In some embodiments, amplified production can be DNA.Right In the case that target RNA is expanded, DNA can be obtained by the reverse transcription of RNA and can be generated using subsequent DNA cloning The DNA product of amplification.The DNA product of amplification can indicate there is target RNA in the biological sample.In the feelings expanded to DNA Under condition, any DNA cloning method as known in the art can be used.The non-limiting example of DNA cloning method includes polymerization Enzyme chain reaction (PCR), PCR mode of texturing (for example, in real time PCR, ApoE gene, assembly PCR, asymmetric pcr, Digital pcr, emulsion-based PCR etc.), helicase dependent PCR, nest-type PRC, heat start PCR, inverse PCR, methylation status of PTEN promoter, Micro- primer PCR (miniprimer PCR), multiplex PCR, nest-type PRC, overlapping-extension PCR, the asymmetric staggeredly PCR of heat (thermal asymmetric interlaced PCR), fall progressively PCR) and ligase chain reaction (LCR).In some feelings Under condition, DNA cloning is linear.In some cases, DNA cloning is exponential.In some cases, DNA cloning uses Nest-type PRC is realized, the sensitivity of the DNA product of detection amplification can be improved

In some cases, the amplification unit can be used or including archaeal dna polymerase.Any suitable DNA can be used Polymerase, including commercially available archaeal dna polymerase.Archaeal dna polymerase is often referred to mix nucleotide in such a way that template combines Enzyme into DNA chain.The non-limiting example of archaeal dna polymerase includes Taq polymerase, Tth polymerase, Tli polymerase, Pfu poly- Synthase, VENT polymerase, DEEPVENT polymerase, EX-Taq polymerase, LA-Taq polymerase, Expand polymerase, Sso polymerization Enzyme, Poc polymerase, Pab polymerase, Mth polymerase, Pho polymerase, ES4 polymerase, Tru polymerase, Tac polymerase, Tne Polymerase, Tma polymerase, Tih polymerase, Tfi polymerase, Platinum Taq polymerase, Hi-Fi polymerase, Tbr polymerization Enzyme, Tfl polymerase, Pfutubo polymerase, Pyrobest polymerase, Pwo polymerase, KOD polymerase, Bst polymerase, Sac are poly- Synthase, Klenow segment and their variant, modification product and derivative.For certain thermal starting polymerase, Ke Nengxu Will at 94 DEG C -95 DEG C 2 minutes to 10 minutes denaturing steps, this may be different according to different polymerases.One In a little situations, the amplification unit can be used or including reverse transcriptase.Reverse transcriptase is often referred to can when in conjunction with RNA template Enzyme nucleotide being incorporated into DNA chain.The non-limiting example of reverse transcriptase includes HIV-1 reverse transcriptase, M-MLV reverse transcription Enzyme, AMV reverse transcriptase, reverse transcriptase of telomere and their variant, modification product and derivative.For example, the DNA Polymerase is Taq polymerase.

In some cases, the amplification unit can be used or including amplification buffer.For example, the amplification buffer can Comprising 50-200mmol/L Tris-HCl buffer (for example, 60-200mmol/L, 70-200mmol/L, 80-200mmol/L, 90-200mmol/L、100-200mmol/L、110-200mmol/L、120-200mmol/L、130-200mmol/L、140- 200mmol/L, 150-200mmol/L, 160-200mmol/L, 170-200mmol/L, 180-200mmol/L or 190- 200mmol/L).The pH value of the amplification buffer can for 6.0-9.0 (for example, about 6.5,7.0,7.5,8.0,8.1,8.2, 8.3,8.4,8.5,8.6,8.7,8.8,8.9 or 9.0 etc.), it can include about 5-30mmol/L Mg in the amplification buffer²⁺(example Such as, 10-30mmol/L Mg²⁺、15-30mmol/L Mg²⁺、20-30mmol/L Mg²⁺、5-15mmol/L Mg²⁺、10-15mmol/ L Mg²⁺Deng).The amplification buffer also may include, such as 300-800mmol/L KCl (such as 400-800mmol/L, 450-800mmol/L, 500-800mmol/L, 550-800mmol/L, 600-800mmol/L or 700-800mmol/L etc.).Example Such as, the amplification buffer can be 10 × LA Taq Buffer II (Mg²⁺Free), it is purchased from TaKaRa company.

In this application, the amplification unit can carry out amplification reaction (for example, PCR reacts), the body of the amplified reaction System can adjust accordingly with the difference of component involved in the amplification step or amplification module, as long as can be effectively It obtains (for example, the amount of the amplified production to carry out reaction acquisition under the conditions of complete ideal is to obtain at least in terms of 100% 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, At least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more It is more) target gene.For example, the amplification reaction system may include in terms of the total reaction volume of 100 μ L: about 10 μ L The Primer composition of amplification buffer, 4 kinds of dNTP each 200 μm of ol/L, 100pmol/L, 2 μ g templates, 2.5 μ g TaqDNA polymerization Enzyme, 1.5mmol/L Mg²⁺With the water (for example, distilled water) for complementing to 100 μ L volumes.

In some cases, the condition of the amplified reaction can correspondingly be adjusted with the length of the target gene It is whole, as long as can effectively obtain (for example, being to carry out the amount of the amplified production of reaction acquisition under the conditions of complete ideal 100% meter, obtains at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, until Few 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more) target gene.For example, in one embodiment, for carrying out PCR reaction, The condition of reaction can be with are as follows: 94 DEG C recycle within 45 seconds within 45 seconds and 55 DEG C within 10 minutes, 94 DEG C 35 times, 72 DEG C 90 seconds, 72 DEG C after 15 minutes It is maintained at 4 DEG C.

The amplification unit also can be used or including material and reagent and/or one or more needed for carrying out PCR amplification Detect the report agent (for example, detectable nucleic acid binding reagents) of amplified production.For example, nucleic acid polymerase, reverse transcriptase, dNTP、Mg²⁺Buffer needed for ion, amplified reaction and/or the report agent etc. for detecting amplified production.

The amplification unit also can be used or expand template needed for the amplified reaction (for example, PCR reacts) including obtaining Material and reagent.For example, the amplification unit can be used or including anxious from B cell acute lymphoblastic leukemia or T cell Property lymphocytic leukemia patient sample of bone marrow in obtain DNA needed for material and reagent.Material needed for the acquisition DNA It may include DNA extraction kit with reagent.For example, the DNA extraction kit can for NucleoBond Kit CB 20, NucleoBond CB 100 or NucleoBond CB 500 etc. (are purchased from NucleoBond company).

In the case where needing decomposition agent, any suitable decomposition agent as known in the art can be used, including commercially available Decomposition agent.The non-limiting example of decomposition agent includes Tris-HCl, EDTA, detergent (for example, Triton X-100, SDS), Lysozyme, glucolase (glucolase), protease E, viral endolysin, outer lysin (exolysin), zymolase (zymolose), lyticase (Iyticase), Proteinase K, endolysin and outer lysin from bacteriophage come from bacteriophage The endolysin of PM2 comes from the endolysin of bacillus subtilis (B.subtilis) bacteriophage PBSX, comes from lactobacillus prophage The endolysin of Lj928, Lj965, bacteriophage 15Phiadh, the endolysin from streptococcus pneumonia bacteriophage Cp-I, agalasisa hammer The bifunctional peptide glycan lysin of bacterium bacteriophage B30, endolysin and outer lysin from prophage bacterium come from Listeria (Listeria) endolysin of bacteriophage, cave albumen (holin)-endolysin, 20 lysis genes of cell, holWMY walsh grape ball Bacterium (Staphylococcus wameri) M bacteriophage varphiWMY, walsh staphylococcus M bacteriophage varphiWMY's Iy5WMY, and combinations thereof.In some cases, buffer may include decomposition agent (for example, lysis buffer).Lysis buffer One example is sodium hydroxide (NaOH).

In this application, term " specific amplification " is typically referred to be combined using the primer, be obtained by PCR reaction Target specificity amplified production.These specific primer combinations can be effectively (for example, to carry out under the conditions of complete ideal The amount for reacting the amplified production obtained is 100% meter, obtains at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, At least 92%, at least 93%, at least 94%, at least 95% or more) the amplification target gene.

In this application, term " primer " is often referred to a bit of single stranded DNA or RNA, (the example in nucleic acid polymerization reaction Such as, in PCR reaction), it can be used as the starting point that polynucleotide chain is extended.Wherein, " reverse primer " is often referred to and coding strand (coding strand) combines the primer of (for example, the nucleotide sequence with coding strand is complementary)." forward primer " is often referred to and mould The primer of plate chain combination.

In this application, term " target gene " typically refer to embody in subject the presence of leukaemia cell and/or The gene of ratio.For example, the target gene may include one or more genes, its segment or its variant selected from the group below: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1.The target gene can be with Including IgH gene rearrangement, IgK gene rearrangement, the gene rearrangement in T cell receptor region etc..

In this application, term " gene rearrangement " typically refers to change gene by rearranging for gene coded sequence Expression product and/or genetic transcription mode.Gene rearrangement can be realized by the fracture and reconnect of DNA molecular.For example, group Polypeptide chain at T cell receptor may include α, β, γ and/or δ peptide chain, and the T cell receptor gene for encoding these polypeptide chains can pass through Gene rearrangement (V-D-J) and so that T cell receptor is generated diversity and/or specificity.

In this application, term " IgH gene rearrangement " typically refers in the gene (IgH) of encoding immune immunoglobulin heavy chain The gene rearrangement (for example, VH-JH and DH-JH gene rearrangement) of generation.

In this application, term " IgK gene rearrangement " typically refers to the gene (IgK) in encoding immune globulin κ light chain The gene rearrangement (for example, V κ-J kappa gene is reset or Kde missing) of middle generation.

In this application, term " acute lymphatic leukemia " (also referred to as acute lymphoblastic Leukemia, or referred to as ALL) it is often referred to the acute hematologic malignancies as caused by internal lymphocyte abnormality proliferation.

In this application, term " B cell acute lymphoblastic leukemia " (B-ALL) is often referred in blood and/or marrow Contain a kind of acute lymphoblastic leukemia caused by excessive B cell lymphoblast (immature white blood cell).

In this application, term " T cell acute lymphoblastic leukemia " (T-ALL) be often referred to blood and/or marrow and/ Or containing one caused by excessive T cell lymphoblast (immature white blood cell) in tissue (for example, vertical phrenic lymph nodes) Kind acute lymphoblastic leukemia.

In this application, term " T cell receptor " (T cell receptor, TCR) is often referred in T cell surface expression Specific receptor.T cell receptor can be responsible for the antigen that identification is presented by major histocompatibility complex (MHC).It is same Antigen may be identified that a certain T cell receptor also can recognize many kinds of antigens by different T cell receptors.T cell receptor can be with CD3 molecule is present in T cell surface in composite form.T cell receptor can be heterodimer, such as by α and β peptide chain group At, or be made of γ and δ peptide chain.

In this application, term " gene rearrangement in T cell receptor region " is typically referred in encoding T cell receptor β subunit Gene (TCRB) in occur gene rearrangement (for example, V β-J β and D β-J β gene rearrangement).For example, the T cell receptor area The gene in domain includes one or more selected from the group below: T cell receptor A, T cell receptor D, T cell receptor B and T cell receptor G。

In this application, term " coding strand " is often referred in double-stranded DNA, is not used in that DNA chain transcribed, should The nucleotide sequence of chain and the transcription sequence of RNA generated are consistent (being with U instead of the T in DNA in RNA), also known as ariyoshi Chain (sense strand).

In this application, term " template strand " is often referred in double-stranded DNA, that DNA chain for being transcribed, also known as Antisense strand (anti-sense strand).

In this application, term " conservative nucleic acid sequence " is often referred to the nucleic acid sequence with high similarity or identity (such as RNA or DNA sequence dna), can be based on (inter-species homologous sequence) between each species or by identical biogenic different molecular Sequence alignment result between (homologous sequence in planting).

In this application, term " subject's specific nucleic acid sequence " be often referred to it is that there is unique base to put in order, From the nucleic acid sequence of particular subject.For example, it can be used for uniquely identifying the subject, or can be used for be originated from should be by The sequence of examination person's (or its tissue, part, cell (such as leukaemia cell)) and other sources (for example, being originated from other subjects) Sequence differentiate.

In this application, term " non-characteristic gene sequence " is often referred to universally present in numerous subjects, not with There is the gene order of obvious relation between persistence in particular subject source.

In this application, term " PCR " is often referred to polymerase chain reaction (Polymerase Chain Reaction), It can be used for expanding specific DNA fragmentation.For example, in PCR reaction, can using the DNA target fragment of quasi- amplification as template, with A pair of is respectively primer with the oligonucleotide fragment of the end template 5' and 3' termini-complementary, in the effect of hot resistant DNA polymerase Under, it is extended up to according to the mechanism of semi-conservative replication along template strand and completes new target fragment synthesis.It can be by repeating this Process and enable target fragment massive amplification.

In this application, term " qPCR " is often referred to quantitative polyase chain reaction, is detected by quantitative nucleic acid amplification Accurate Analysis is carried out to the quantity of target nucleic acid molecules.

In this application, term " sequencing " is often referred to the composition of the sequence for measuring target molecule (for example, nucleic acid molecules) Technology, can be used for determining the specific nucleotide sequence of target nucleic acid molecules.Technology currently used for sequencing mainly has Sanger The chemical degradation method of the invention such as the double deoxidating chain end cessation method, the Maxam that are invented Deng (1977) (1977) and Taq cycle sequencing Method etc..

In this application, 1/7 extra that primer " VH1/7 " is often referred to specifically bind and/or expand the area IgH gene V is shown The primer of son.For example, the gene region of primer targeting is VH1/7 exon.

In this application, primer " JH cons " is often referred to specifically bind and/or the amplification area IgH gene J is shared (concensus) primer of Sequence.For example, the gene region of primer targeting is JH concensus.

In this application, primer " VH2 " is often referred to 2 exons for specifically binding and/or expanding the area IgH gene V Primer.For example, the gene region of primer targeting is VH2 exon.

In this application, primer " VH3 " is often referred to 3 exons for specifically binding and/or expanding the area IgH gene V Primer.For example, the gene region of primer targeting is VH3 exon.

In this application, primer " VH4 " is often referred to 4 exons for specifically binding and/or expanding the area IgH gene V Primer.For example, the gene region of primer targeting is VH4 exon.

In this application, primer " VH5 " is often referred to 5 exons for specifically binding and/or expanding the area IgH gene V Primer.For example, the gene region of primer targeting is VH5 exon.

In this application, primer " VH6 " is often referred to 6 exons for specifically binding and/or expanding the area IgH gene V Primer.For example, the gene region of primer targeting is VH6 exon.

In this application, primer " DH1 " is often referred to 1 exon for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH1 exon.

In this application, primer " DH2 " is often referred to 2 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH2 exon.

In this application, primer " DH3 " is often referred to 3 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH3 exon.

In this application, primer " DH4 " is often referred to 4 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH4 exon.

In this application, primer " DH5 " is often referred to 5 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH5 exon.

In this application, primer " DH6 " is often referred to 6 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH6 exon.

In this application, primer " DH7 " is often referred to 7 exons for specifically binding and/or expanding the area IgH gene D Primer.For example, the gene region of primer targeting is DH7 exon.

In this application, primer " Vk1 " is often referred to 1 exon for specifically binding and/or expanding the area IgK gene V Primer.For example, the gene region of primer targeting is Vk1 exon.

In this application, primer " Vk2 " is often referred to 2 exons for specifically binding and/or expanding the area IgK gene V Primer.For example, the gene region of primer targeting is Vk2 exon.

In this application, primer " Vk3 " is often referred to 3 exons for specifically binding and/or expanding the area IgK gene V Primer.For example, the gene region of primer targeting is Vk3 exon.

In this application, primer " Kdel " is often referred to specifically bind and/or expand the kappa missing element of IgK gene The primer of (kappa deleting element).For example, the gene region of primer targeting is the region Kdel.

In this application, primer " Intron RSS " is often referred to specifically bind and/or expand the introne weight of IgK gene The primer of group signal sequence (intron recombination signal sequence).For example, the gene of primer targeting Region is the region Intron RSS of IgK.

In this application, primer " Vd2 " is often referred to specifically bind and/or expand the area TCR Delta (TCR δ) gene V The primer of 2 exons.For example, the gene region of primer targeting is V δ 2 (Vd2) exon.

In this application, primer " Dd3 " is often referred to 3 exons for specifically binding and/or expanding the area TCR δ gene D Primer.For example, the gene region of primer targeting is D δ 3 (Dd3) exon.

In this application, primer " Dd2 " is often referred to 2 exons for specifically binding and/or expanding the area TCR δ gene D Primer.For example, the gene region of primer targeting is D δ 2 (Dd2) exon.

In this application, primer " Ja29 " is often referred to specifically bind and/or expand the area TCR Alpha (TCR α) gene J 29 exons primer.For example, the gene region of primer targeting is J α 29 (Ja29) exon.

In this application, primer " Ja9 " is often referred to 9 exons for specifically binding and/or expanding the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 9 (Ja9) exon.

In this application, primer " Ja30 " is often referred to specifically bind and/or expand 30 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 30 (Ja30) exon.

In this application, primer " Ja48 " is often referred to specifically bind and/or expand 48 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 48 (Ja48) exon.

In this application, primer " Ja49 " is often referred to specifically bind and/or expand 49 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 49 (Ja49) exon.

In this application, primer " Ja52 " is often referred to specifically bind and/or expand 52 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 52 (Ja52) exon.

In this application, primer " Ja54 " is often referred to specifically bind and/or expand 54 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 54 (Ja54) exon.

In this application, primer " Ja55 " is often referred to specifically bind and/or expand 55 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 55 (Ja55) exon.

In this application, primer " Ja56 " is often referred to specifically bind and/or expand 56 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 56 (Ja56) exon.

In this application, primer " Ja57 " is often referred to specifically bind and/or expand 57 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 57 (Ja57) exon.

In this application, primer " Ja58 " is often referred to specifically bind and/or expand 58 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 58 (Ja58) exon.

In this application, primer " Ja59 " is often referred to specifically bind and/or expand 59 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 59 (Ja59) exon.

In this application, primer " Ja61 " is often referred to specifically bind and/or expand 61 exons in the area TCR α gene J Primer.For example, the gene region of primer targeting is J α 61 (Ja61) exon.

In this application, primer " Vg1 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene V The primer of 1 exon.For example, the gene region of primer targeting is Vg1 exon.

In this application, primer " Vg2 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene V The primer of 2 exons.For example, the gene region of primer targeting is Vg2 exon.

In this application, primer " Vg4 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene V The primer of 4 exons.For example, the gene region of primer targeting is Vg4 exon.

In this application, primer " Jg1 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene J The primer of 1 exon.For example, the gene region of primer targeting is Jg1 exon.

In this application, primer " Jg2 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene J The primer of 2 exons.For example, the gene region of primer targeting is Jg2 exon.

In this application, primer " Vd1 " is often referred to specifically bind and/or expand the area TCR Delta (TCR δ) gene V The primer of 1 exon.For example, the gene region of primer targeting is V δ 1 (Vd1) exon.

In this application, primer " Jd1 " is often referred to specifically bind and/or expand the area TCR Delta (TCR δ) gene J The primer of 1 exon.For example, the gene region of primer targeting is J δ 1 (Jd1) exon.

In this application, primer " Vd3 " is often referred to specifically bind and/or expand the area TCR Delta (TCR δ) gene V The primer of 3 exons.For example, the gene region of primer targeting is V δ 3 (Vd3) exon.

In this application, primer " Vg3 " is often referred to specifically bind and/or expand the area TCR Gamma (TCRG) gene V The primer of 3 exons.For example, the gene region of primer targeting is Vg3 exon.

In this application, primer " Sildb " is often referred to specifically bind and/or amplification sil gene disruption splits site (breakpoint) primer.For example, the gene region of primer targeting is sil broken site.

In this application, primer " Taldb1 " is often referred to specifically bind and/or amplification tal-1 gene disruption splits site 1 (breakpoint1) primer.For example, the gene region of primer targeting is tal-1 broken site 1.

In this application, primer " Vb2 " is often referred to 2 for specifically binding and/or expanding the area TCR Beta (TCR β) gene V The primer of exon.For example, the gene region of primer targeting is V β 2 (Vb2) exon.

In this application, primer " Vb4 " is often referred to 4 for specifically binding and/or expanding the area TCR Beta (TCR β) gene V The primer of exon.For example, the gene region of primer targeting is V β 4 (Vb4) exon.

In this application, primer " Vb5/1 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V 5/1 exon primer.For example, the gene region of primer targeting is V β 5/1 (Vb5/1) exon.

In this application, primer " Vb6a/11 " is often referred to specifically bind and/or expand TCR Beta (TCR β) gene V The primer of the 6a/11 exon in area.For example, the gene region of primer targeting is V β 6a/11 (Vb6a/11) exon.

In this application, primer " Vb6b/25 " is often referred to specifically bind and/or expand TCR Beta (TCR β) gene V The primer of the 6b/25 exon in area.For example, the gene region of primer targeting is V β 6b/25 (Vb6b/25) exon.

In this application, primer " Vb6c " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 6c exon.For example, the gene region of primer targeting is V β 6c (Vb6c) exon.

In this application, primer " Vb7 " is often referred to 7 for specifically binding and/or expanding the area TCR Beta (TCR β) gene V The primer of exon.For example, the gene region of primer targeting is V β 7 (Vb7) exon.

In this application, primer " Vb8a " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 8a exon.For example, the gene region of primer targeting is V β 8a (Vb8a) exon.

In this application, primer " Vb9 " is often referred to 9 for specifically binding and/or expanding the area TCR Beta (TCR β) gene V The primer of exon.For example, the gene region of primer targeting is V β 9 (Vb9) exon.

In this application, primer " Vb10 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 10 exons.For example, the gene region of primer targeting is V β 10 (Vb10) exon.

In this application, primer " Vb11 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 11 exons.For example, the gene region of primer targeting is V β 11 (Vb11) exon.

In this application, primer " Vb12a/3/13a/15 " is often referred to specifically bind and/or expand TCR Beta (TCR β) the primer of the 12a/3/13a/15 exon in the area gene V.For example, the gene region of primer targeting is V β 12a/3/13a/ 15 (Vb12a/3/13a/15) exons.

In this application, primer " Vb13b " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V 13b exon primer.For example, the gene region of primer targeting is V β 13b (Vb13b) exon.

In this application, primer " Vb13c/12b/14 " is often referred to specifically bind and/or expands TCR Beta (TCR β) The primer of the 13c/12b/14 exon in the area gene V.For example, the gene region of primer targeting is V β 13c/12b/14 (Vb13c/12b/14) exon.

In this application, primer " Vb16 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 16 exons.For example, the gene region of primer targeting is V β 16 (Vb16) exon.

In this application, primer " Vb17 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 17 exons.For example, the gene region of primer targeting is V β 17 (Vb17) exon.

In this application, primer " Vb18 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 18 exons.For example, the gene region of primer targeting is V β 18 (Vb18) exon.

In this application, primer " Vb19 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 19 exons.For example, the gene region of primer targeting is V β 19 (Vb19) exon.

In this application, primer " Vb20 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 20 exons.For example, the gene region of primer targeting is V β 20 (Vb20) exon.

In this application, primer " Vb21 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 21 exons.For example, the gene region of primer targeting is V β 21 (Vb21) exon.

In this application, primer " Vb22 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 22 exons.For example, the gene region of primer targeting is V β 22 (Vb22) exon.

In this application, primer " Vb23/8b " is often referred to specifically bind and/or expand TCR Beta (TCR β) gene V The primer of the 23/8b exon in area.For example, the gene region of primer targeting is V β 23/8b (Vb23/8b) exon.

In this application, primer " Vb24 " is often referred to specifically bind and/or expand the area TCR Beta (TCR β) gene V The primer of 24 exons.For example, the gene region of primer targeting is V β 24 (Vb24) exon.

In this application, primer " Db1 " is often referred to 1 for specifically binding and/or expanding the area TCR Beta (TCR β) gene D The primer of exon.For example, the gene region of primer targeting is D β 1 (Db1) exon.

In this application, primer " Db2 " is often referred to 2 for specifically binding and/or expanding the area TCR Beta (TCR β) gene D The primer of exon.For example, the gene region of primer targeting is D β 2 (Db2) exon.

In this application, primer " Jb1 " is often referred to 1 for specifically binding and/or expanding the area TCR Beta (TCR β) gene J The primer of exon.For example, the gene region of primer targeting is J β 1 (Jb1) exon.

In this application, primer " Jb2 " is often referred to 2 for specifically binding and/or expanding the area TCR Beta (TCR β) gene J The primer of exon.For example, the gene region of primer targeting is J β 2 (Jb2) exon.

For example, the reagent for capableing of specific amplification target gene (IgH gene rearrangement) includes selected from following one group Or multiple groups primer combination: 1) forward primer: VH1/7, reverse primer: JH cons；2) forward primer: VH2, reverse primer: JH cons；3) forward primer: VH3, reverse primer: JH cons；4) forward primer: VH4, reverse primer: JH cons；5) forward direction is drawn Object: VH5, reverse primer: JH cons；6) forward primer: VH6, reverse primer: JH cons；7)

Forward primer: DH1, DH4, DH5 and DH7, reverse primer: JH cons；8) forward primer: DH2, reverse primer: JH cons；9) forward primer: DH3, reverse primer: JH cons；With 10) forward primer: DH6, reverse primer: JH cons.Wherein The primer JH cons includes sequence selected from the group below: sequence shown in SEQ ID NO.19；With shown in SEQ ID NO.20 Sequence.The primer VH1/7 includes sequence selected from the group below: SEQ ID NO.1 is shown, SEQ ID NO.2 is shown and SEQ ID Sequence shown in NO.3.The primer VH2 includes sequence selected from the group below: shown in SEQ ID NO.4, shown in SEQ ID NO.5 With sequence shown in SEQ ID NO.6.The primer VH3 includes sequence selected from the group below: shown in SEQ ID NO.7, SEQ ID Shown in NO.8 and sequence shown in SEQ ID NO.9.The primer VH4 includes sequence selected from the group below: SEQ ID NO.10 institute Show, shown in SEQ ID NO.11 and sequence shown in SEQ ID NO.12.The primer VH5 includes sequence selected from the group below: SEQ Shown in ID NO.13, shown in SEQ ID NO.14 and sequence shown in SEQ ID NO.15.The primer VH6 includes to be selected from the group Sequence: shown in SEQ ID NO.16, SEQ ID NO.17 and sequence shown in SEQ ID NO.18.The primer DH1 includes Sequence selected from the group below: shown in SEQ ID NO.21 and sequence shown in SEQ ID NO.22.The primer DH2 includes to be selected from down The sequence of group: shown in SEQ ID NO.23 and sequence shown in SEQ ID NO.24.The primer DH3 includes sequence selected from the group below Column: shown in SEQ ID NO.25 and sequence shown in SEQ ID NO.26.The primer DH4 includes sequence selected from the group below: SEQ Shown in ID NO.27 and sequence shown in SEQ ID NO.28.The primer DH5 includes sequence selected from the group below: SEQ ID Shown in NO.29 and sequence shown in SEQ ID NO.30.The primer DH6 includes sequence selected from the group below: SEQ ID NO.31 Sequence shown in shown and SEQ ID NO.32.The primer DH7 includes sequence selected from the group below: shown in SEQ ID NO.33 and Sequence shown in SEQ ID NO.34.

For example, the reagent for capableing of specific amplification target gene (IgK gene rearrangement) includes selected from following one group Or multiple groups primer combination: 1) forward primer: Vk1, reverse primer: Kdel；2) forward primer: Vk2, reverse primer: Kdel；3) just To primer: Vk3, reverse primer: Kdel；With 4) forward primer: Intron RSS, reverse primer: Kdel.The wherein primer Vk1 includes sequence selected from the group below: shown in SEQ ID NO.35 and sequence shown in SEQ ID NO.36.The primer Vk2 packet Containing sequence selected from the group below: shown in SEQ ID NO.37 and sequence shown in SEQ ID NO.38.The primer Vk3 includes to be selected from The sequence of the following group: shown in SEQ ID NO.39 and sequence shown in SEQ ID NO.40.The primer Vk4 includes selected from the group below Sequence: shown in SEQ ID NO.41 and sequence shown in SEQ ID NO.42.The primer I ntron RSS includes to be selected from the group Sequence: shown in SEQ ID NO.43 and sequence shown in SEQ ID NO.44.The primer Kdel includes sequence selected from the group below Column: shown in SEQ ID NO.45 and sequence shown in SEQ ID NO.46.

For example, be directed to B cell acute lymphoblastic leukemia, it is described being capable of specific amplification target gene (T cell receptor Regional gene is reset) reagent include being combined selected from following one or more groups of primers: 1) forward primer: Vd2, reverse primer: Dd3；2) forward primer: Dd2, reverse primer: Dd3；3) forward primer: Vd2, reverse primer: Ja29；4)

Forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；5) forward primer: Vg1, reverse primer: Jg1 and Jg2；6) forward primer: Vg2 reversely draws Object: Jg1 and Jg2；7) forward primer: Vg4, reverse primer: Jg1 and Jg2；8) the more primers of TCRB combine A；9) the more primer sets of TCRB Close B；C is combined with the more primers of 10) TCRB.Wherein the primer Vg1 includes sequence selected from the group below: shown in SEQ ID NO.47 and Sequence shown in SEQ ID NO.48.The primer Vg2 includes sequence selected from the group below: shown in SEQ ID NO.49 and SEQ ID Sequence shown in NO.50.The primer Vg4 includes sequence selected from the group below: shown in SEQ ID NO.53 and SEQ ID NO.54 Shown in sequence.The primer Jg1 includes sequence selected from the group below: shown in SEQ ID NO.55 and shown in SEQ ID NO.56 Sequence.The primer Jg2 includes sequence selected from the group below: shown in SEQ ID NO.57 and sequence shown in SEQ ID NO.58. The primer Vd2 includes sequence selected from the group below: shown in SEQ ID NO.61 and sequence shown in SEQ ID NO.62.It is described to draw Object Dd2 includes sequence selected from the group below: shown in SEQ ID NO.65 and sequence shown in SEQ ID NO.66.The primer Dd3 Include sequence selected from the group below: shown in SEQ ID NO.67 and sequence shown in SEQ ID NO.68.The primer Ja9 includes choosing From the sequence of the following group: sequence shown in SEQ ID NO.71.The primer Ja29 includes sequence selected from the group below: SEQ ID Sequence shown in NO.72.The primer Ja30 includes sequence selected from the group below: sequence shown in SEQ ID NO.73.It is described to draw Object Ja48 includes sequence selected from the group below: sequence shown in SEQ ID NO.74.The primer Ja49 includes sequence selected from the group below Column: sequence shown in SEQ ID NO.75.The primer Ja52 includes sequence selected from the group below: sequence shown in SEQ ID NO.76 Column.The primer Ja54 includes sequence selected from the group below: sequence shown in SEQ ID NO.77.The primer Ja55 includes to be selected from The sequence of the following group: sequence shown in SEQ ID NO.78.The primer Ja56 includes sequence selected from the group below: SEQ ID NO.79 Shown in sequence.The primer Ja57 includes sequence selected from the group below: sequence shown in SEQ ID NO.80.The primer Ja58 Include sequence selected from the group below: sequence shown in SEQ ID NO.81.The primer Ja59 includes sequence selected from the group below: SEQ Sequence shown in ID NO.82.The primer Ja61 includes sequence selected from the group below: sequence shown in SEQ ID NO.83.It is described More primer combination A include one or more sequences selected from the group below: SEQ ID NO.86 is shown, SEQ ID NO.87 is shown, SEQ Shown in sequence shown in ID NO.88, SEQ ID NO.89, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID Shown in NO.92, SEQ ID NO.93 is shown, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID NO.96 institute Show, SEQ ID NO.97 is shown, SEQ ID NO.98 is shown, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ Shown in ID NO.101, SEQ ID NO.102 is shown, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID Shown in NO.105, SEQ ID NO.106 is shown, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID Shown in NO.111, SEQ ID NO.112 is shown, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID Shown in NO.115, SEQ ID NO.116 is shown, SEQ ID NO.118, SEQ ID NO.122 are shown and SEQ ID NO.123 institute The sequence shown.The more primers combination B includes one or more sequences selected from the group below: shown in SEQ ID NO.86, SEQ ID Shown in NO.87, SEQ ID NO.88 is shown, SEQ ID NO.89 is shown, SEQ ID NO.90 is shown, SEQ ID NO.91 institute Show, SEQ ID NO.92 is shown, SEQ ID NO.93 is shown, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID Shown in NO.96, SEQ ID NO.97 is shown, SEQ ID NO.98 is shown, SEQ ID NO.99 is shown, SEQ ID NO.100 institute Show, shown in SEQ ID NO.101, shown in SEQ ID NO.102, shown in SEQ ID NO.103, shown in SEQ ID NO.104, Shown in SEQ ID NO.105, SEQ ID NO.106 is shown, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ Shown in ID NO.117, shown in SEQ ID NO.119, shown in SEQ ID NO.120 and sequence shown in SEQ ID NO.121.Institute Stating more primers combination C includes one or more sequences selected from the group below: shown in SEQ ID NO.109, SEQ ID NO.110 institute Show, shown in SEQ ID NO.111, shown in SEQ ID NO.112, shown in SEQ ID NO.113, shown in SEQ ID NO.114, Shown in SEQ ID NO.115, SEQ ID NO.116 is shown, SEQ ID NO.117 is shown, SEQ ID NO.118 is shown, SEQ Shown in ID NO.119, SEQ ID NO.120 is shown, SEQ ID NO.121 is shown, SEQ ID NO.122 is shown and SEQ ID Sequence shown in NO.123.

For example, be directed to T cell acute lymphoblastic leukemia, it is described being capable of specific amplification target gene (T cell receptor Regional gene is reset) reagent include being combined selected from following one or more groups of primers: 1) forward primer: Vd1, reverse primer: Jd1；2) forward primer: Vd2, reverse primer: Jd1；3) forward primer: Vd3, reverse primer: Jd1；4) forward primer: Vd2, instead To primer: Dd3；5) forward primer: Dd2, reverse primer: Dd3；6) forward primer: Dd2, reverse primer: Jd1；7) forward direction is drawn Object: Vd2, reverse primer: Ja29；8) forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；9) forward primer: Vg1, reverse primer: Jg1 and Jg2；10) positive Primer: Vg2, reverse primer: Jg1 and Jg2；11)

Forward primer: Vg3, reverse primer: Jg1 and Jg2；12) forward primer: Vg4, reverse primer: Jg1 and Jg2；13)

Forward primer: Sildb, reverse primer: Taldb1；14) the more primers of TCRB combine A；15) the more primers of TCRB combine B； C is combined with the more primers of 16) TCRB.Wherein the primer Vg3 includes sequence selected from the group below: shown in SEQ ID NO.51 and SEQ Sequence shown in ID NO.52.The primer Vd3 includes sequence selected from the group below: shown in SEQ ID NO.63 and SEQ ID Sequence shown in NO.64.The primer Vg1 includes sequence selected from the group below: shown in SEQ ID NO.47 and SEQ ID NO.48 Shown in sequence.The primer Vg2 includes sequence selected from the group below: shown in SEQ ID NO.49 and shown in SEQ ID NO.50 Sequence.The primer Vg4 includes sequence selected from the group below: shown in SEQ ID NO.53 and sequence shown in SEQ ID NO.54. The primer Jg1 includes sequence selected from the group below: shown in SEQ ID NO.55 and sequence shown in SEQ ID NO.56.It is described to draw Object Jg2 includes sequence selected from the group below: shown in SEQ ID NO.57 and sequence shown in SEQ ID NO.58.The primer Vd2 Include sequence selected from the group below: shown in SEQ ID NO.61 and sequence shown in SEQ ID NO.62.The primer Dd2 includes choosing From the sequence of the following group: shown in SEQ ID NO.65 and sequence shown in SEQ ID NO.66.The primer Dd3 includes to be selected from the group Sequence: shown in SEQ ID NO.67 and sequence shown in SEQ ID NO.68.The primer Ja9 includes sequence selected from the group below Column: sequence shown in SEQ ID NO.71.The primer Ja29 includes sequence selected from the group below: sequence shown in SEQ ID NO.72 Column.The primer Ja30 includes sequence selected from the group below: sequence shown in SEQ ID NO.73.The primer Ja48 includes to be selected from The sequence of the following group: sequence shown in SEQ ID NO.74.The primer Ja49 includes sequence selected from the group below: SEQ ID NO.75 Shown in sequence.The primer Ja52 includes sequence selected from the group below: sequence shown in SEQ ID NO.76.The primer Ja54 Include sequence selected from the group below: sequence shown in SEQ ID NO.77.The primer Ja55 includes sequence selected from the group below: SEQ Sequence shown in ID NO.78.The primer Ja56 includes sequence selected from the group below: sequence shown in SEQ ID NO.79.It is described Primer Ja57 includes sequence selected from the group below: sequence shown in SEQ ID NO.80.The primer Ja58 includes selected from the group below Sequence: sequence shown in SEQ ID NO.81.The primer Ja59 includes sequence selected from the group below: shown in SEQ ID NO.82 Sequence.The primer Ja61 includes sequence selected from the group below: sequence shown in SEQ ID NO.83.More primers combine A packet Containing one or more sequences selected from the group below: SEQ ID NO.86 is shown, SEQ ID NO.87 is shown, SEQ ID NO.88 institute Show, SEQ ID NO.89 is shown, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID Shown in NO.93, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 institute Show, SEQ ID NO.98 is shown, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ Shown in ID NO.102, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID NO.105 is shown, SEQ ID Shown in NO.106, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID NO.111 is shown, SEQ ID Shown in NO.112, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ ID Shown in NO.116, shown in SEQ ID NO.118, shown in SEQ ID NO.122 and sequence shown in SEQ ID NO.123.It is described More primer combination B include one or more sequences selected from the group below: SEQ ID NO.86 is shown, SEQ ID NO.87 is shown, SEQ Shown in ID NO.88, SEQ ID NO.89 is shown, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 Shown in shown, SEQ ID NO.93, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ Shown in ID NO.97, SEQ ID NO.98 is shown, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID Shown in NO.101, SEQ ID NO.102 is shown, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID Shown in NO.105, SEQ ID NO.106 is shown, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID Shown in NO.117, shown in SEQ ID NO.119, shown in SEQ ID NO.120 and sequence shown in SEQ ID NO.121.It is described More primer combination C include one or more sequences selected from the group below: SEQ ID NO.109 is shown, SEQ ID NO.110 is shown, Shown in SEQ ID NO.111, SEQ ID NO.112 is shown, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ Shown in ID NO.115, SEQ ID NO.116 is shown, SEQ ID NO.117 is shown, SEQ ID NO.118 is shown, SEQ ID Shown in NO.119, SEQ ID NO.120 is shown, SEQ ID NO.121 is shown, SEQ ID NO.122 is shown and SEQ ID Sequence shown in NO.123.The primer Vd1 includes sequence selected from the group below: shown in SEQ ID NO.59 and SEQ ID NO.60 Shown in sequence.The primer Jd1 includes sequence selected from the group below: shown in SEQ ID NO.69 and shown in SEQ ID NO.70 Sequence.The primer Sildb includes sequence selected from the group below: sequence shown in SEQ ID NO.84.The primer Taldb1 packet Containing sequence selected from the group below: sequence shown in SEQ ID NO.85.

In this application, term " identification unit " is often referred to identify is originated from the target specificity amplified production The functional unit of the characteristic amplified production of the leukaemia cell of the subject, the identification unit may include for identifying State the reagent and device of target specificity amplified production.The identification unit can effectively (for example, at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% Or more) will be originated from the subject leukaemia cell characteristic amplified production from the target specificity amplified production It separates.For example, the characteristic amplified production from subject leukaemia cell can be separated by gel electrophoresis.It is identifying In the process, isolated product is (for example, target is that the isolated characteristic amplification from subject leukaemia cell produces Object) specific location that can be displayed in gel in gel electrophoresis, by by itself and nucleic acid fragment corresponding in positive controls Be compared (for example, the two be length about 400pb nucleic acid fragment), can tentatively judge through the identification module separate The product (for example, DNA fragmentation) of acquisition is the characteristic amplified production from the subject leukaemia cell.

Gel electrophoresis separation can be by the characteristic amplified production of the leukaemia cell of the subject effectively (example Such as, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% or More) separated from the target specificity amplified production obtained in the amplification step or amplification module.The mirror Order member contains for reagent and device needed for carrying out gel electrophoresis separation, for example, for carrying out agarose gel electrophoresis Or the reagent and device of polyacrylamide gel electrophoresis.Such as the identification unit may include reagent needed for preparing gel And/or electrophoretic buffer.For example, reagent needed for the preparation gel may include agarose, acrylamide (Acr), N, N- is sub- One of first bisacrylamide (Bis), tetramethylethylenediamine (TEMED) and trishydroxymethylaminomethane (Tris) are a variety of. For example, using 2% (w/w) Ago-Gel.

The electrophoretic buffer can containing Tris-HCl, dithiothreitol (DTT) (DTT), SDS, bromophenol blue, beta -mercaptoethanol and One of glycerol is a variety of.For example, the electrophoretic buffer includes 100mmol/L Tris-HCl (6.8), 200mmol/L bis- Sulphur threitol (DTT), 4%SDS, 0.2% bromophenol blue and 20% glycerol.For example, in one embodiment, the running buffer Liquid contain 60mmol/L Tris-HCl (pH6.8), 2%SDS, 0.1% bromophenol blue, 25% glycerol and 14.4mmol/L β-sulfydryl Ethyl alcohol.For another example, the identification unit may also include electrophoresis tank and/or electrophoresis apparatus.For example, the electrophoresis tank can be disk electricity Swimming slot, vertical version electrophoresis tank or Horizontal electrophoresis tank.The electrophoresis apparatus can be voltage stabilization and current stabilization electrophoresis apparatus, full-automatic fluorescence/visible Light dual system electrophoresis apparatus or full-automatic agarose electrophoresis instrument.The identification unit may also include plastic plate, power supply etc. and be convenient for The attachment device of gel electrophoresis separation.The condition of the gel electrophoresis separation can be according to separated nucleic acid (for example, DNA) piece Section length and accordingly adjust, only isolated product segment (for example, DNA fragmentation) band need to be made clear, meet it is above-mentioned effectively Isolated requirement.The condition of the gel electrophoresis separation can be persistently to be powered at voltage 100V 2-3 hours；Or To be persistently powered overnight at voltage 40-50V.

In this application, term " analytical unit " typically refers to obtain the spy for the leukaemia cell for being specific to the subject The functional unit of sign property gene order, the analytical unit may include the reagent and dress for analyzing the characteristic amplified production It sets.

The analytical unit can be accurately (for example, gene order and true base by obtaining the analytical unit Because sequence is compared, sequence similarity reaches at least 85%, at least 86%, at least 87%, at least 88%, at least 89%, until Few 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99% or more) the characteristic gene sequence of the leukaemia cell of the subject is obtained.

In this application, term " characteristic gene sequence " typically refers to there is unique base to put in order, source In the nucleic acid sequence of particular subject.For example, it can be used for uniquely identifying the subject, or can be used for that the subject will be originated from The sequence of the sequence of (or its tissue, part, cell (such as leukaemia cell)) and other sources (for example, being originated from other subjects) Column differentiate.

In this application, term " characteristic amplified production " typically refers to the amplified production of reflection subject's specificity.Example Such as, reflection is specific to the amplified production of the characteristic gene sequence of the subject leukaemia cell.

In this application, term " nucleic acid sequencing " typically refers to the skill of the composition of the sequence for measuring target nucleic acid molecules Art can be used for determining the specific nucleotide sequence of target nucleic acid molecules.Technology currently used for sequencing mainly has Sanger etc. (1977) chemical degradation method and Taq cycle PCR sequencing PCR of the invention such as the double deoxidating chain end cessation method, the Maxam that invent (1977) Deng.

The analytical unit may include examination needed for carrying out nucleic acid sequencing and sequence analysis to the characteristic amplified production Agent and device.For example, the analytical unit may include implementing PCR sequencing PCR, Taq cycle PCR sequencing PCR or SILVER SEQUENCETM Reagent needed for DNA sequencing system method and device.When using Taq cycle PCR sequencing PCR, the analytical unit may include general Positive sequencing primer and Ready Reaction Dye Deoxy Terminator Cycle sequencing kit (ABI company).Or Person can be used the analytical unit and analyze the characteristic amplified production of the leukaemia cell of the subject, specific Step can be found in Kneba M, Bolz I, Linke B, Hiddemann W.Analysis of rearranged T-cell receptor beta-chain genes by polymerase chain reaction(PCR)DNA sequencing and automated high resolution PCR fragment analysis.Blood 1995；In 86:3930-3937 The record of the part " Cloning and DNA sequencing " or referring to document Linke B, Bolz I, Fayyazi A, von Hofen M,Pott C,Bertram J et al.Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.Leukemia 1997；The record of the part " Cloning and DNA sequencing " in 11:1055-1062., this The full text of a little documents is cited in the application.

In this application, term " sequence analysis " typically refers to analyze by the result to the nucleic acid sequencing, can Specify the characteristic gene sequence whether resulting nucleic acid sequencing result belongs to the subject leukaemia cell.

The analytical unit may include the reagent and device analyzed the result of the nucleic acid sequencing, such as may include Needed for progress BLAST (Basic Local Alignment Search Tool), BioEdit software or the analysis of MEGA software Equipment and device.Such as to the analysis module input respectively the nucleic acid sequencing result and be prevalent in arbitrarily by The non-characteristic gene order of examination person obtains the characteristic gene sequence for characterizing the subject leukaemia cell by analysis Column.

In this application, term " recognition unit " typically refers to judge the presence of residual leukemic cell in the subject And/or the functional unit of ratio.The recognition unit may include be capable of specific recognition and/or the amplification subject described The reagent and device of characteristic gene sequence, to judge the presence and/or ratio of residual leukemic cell in the subject. For example, the recognition unit can effectively obtain (for example, to carry out the amount of the product of reaction acquisition under the conditions of complete ideal For 100% meter, at least 75%, at least 80%, at least 81%, at least 82%, at least 83%, at least 84%, at least 85% is obtained, At least 86%, at least 87%, at least 88%, at least 89%, at least 90%, at least 91%, at least 92%, at least 93%, at least 94%, at least 95% or more) it is specific to the characteristic gene sequence of the subject leukaemia cell.

The recognition unit may include the characteristic gene sequence that specific amplification is specific to the subject leukaemia cell The reagent or device of column.

The recognition unit may include probe in conjunction with non-characteristic gene sequence and can expand non-characteristic base Because of the reagent or device of sequence.In this application, term " non-characteristic gene sequence " is typically referred to universally present in numerous In subject, there is not the gene order of obvious relation between persistence with particular subject source.

For example, the non-characteristic gene sequence may include the nucleic acid sequence from one of the following group or several genes: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1.The wherein non-characteristic base Because sequence includes the nucleic acid sequence from IgH, and the reagent that can expand non-characteristic gene sequence or device include choosing From the sequence of the following group: shown in SEQ ID NO.124, SEQ ID NO.125 is shown, SEQ ID NO.126 is shown, SEQ ID Shown in NO.127, SEQ ID NO.128 is shown, SEQ ID NO.129 is shown, SEQ ID NO.130 is shown and SEQ ID Sequence shown in NO.131.The non-characteristic gene sequence includes the nucleic acid sequence from IgH, and described with non-characteristic base Because the probe that sequence combines includes sequence selected from the group below: SEQ ID NO.173 is shown, SEQ ID NO.174 is shown, SEQ ID Shown in NO.175, SEQ ID NO.176 is shown, SEQ ID NO.177 is shown, SEQ ID NO.178 is shown, SEQ ID Shown in NO.179 and sequence shown in SEQ ID NO.180.The non-characteristic gene sequence includes the nucleic acid sequence from IgK Column, and the reagent that can expand non-characteristic gene sequence or device include sequence selected from the group below: SEQ ID NO.132 Shown in shown, SEQ ID NO.133, shown in SEQ ID NO.134, shown in SEQ ID NO.135, shown in SEQ ID NO.136, Shown in SEQ ID NO.137, SEQ ID NO.138 is shown, SEQ ID NO.139 is shown, SEQ ID NO.140 is shown and SEQ Sequence shown in ID NO.141.The non-characteristic gene sequence includes the nucleic acid sequence from IgK, and described with non-feature Property the probe that combines of gene order include sequence selected from the group below: shown in SEQ ID NO.181, shown in SEQ ID NO.182, Shown in SEQ ID NO.183, SEQ ID NO.184 is shown, SEQ ID NO.185 is shown, SEQ ID NO.186 is shown, SEQ Shown in ID NO.187, shown in SEQ ID NO.188, shown in SEQ ID NO.189 and sequence shown in SEQ ID NO.190.Institute Stating non-characteristic gene sequence includes the nucleic acid sequence from T cell receptor G, and described can expand non-characteristic gene sequence Reagent or device include sequence selected from the group below: shown in SEQ ID NO.142, shown in SEQ ID NO.143, SEQ ID Shown in NO.144, SEQ ID NO.145 is shown, SEQ ID NO.146 is shown, SEQ ID NO.147 is shown, SEQ ID Shown in NO.148, shown in SEQ ID NO.149, shown in SEQ ID NO.150 and sequence shown in SEQ ID NO.151.It is described Non- characteristic gene sequence includes the nucleic acid sequence from T cell receptor G, and the spy in conjunction with non-characteristic gene sequence Needle includes sequence selected from the group below: SEQ ID NO.191 is shown, SEQ ID NO.192 is shown, SEQ ID NO.193 is shown, Shown in SEQ ID NO.194, SEQ ID NO.195 is shown, SEQ ID NO.196 is shown, SEQ ID NO.197 is shown, SEQ Shown in ID NO.198, shown in SEQ ID NO.199 and sequence shown in SEQ ID NO.200.The non-characteristic gene sequence Nucleic acid sequence including being originated from T cell receptor D and/or T cell receptor A, and described can expand non-characteristic gene sequence Reagent or device include sequence selected from the group below: SEQ ID NO.152 is shown, SEQ ID NO.153 is shown, SEQ ID Shown in NO.154, shown in SEQ ID NO.155, shown in SEQ ID NO.156 and sequence shown in SEQ ID NO.157.It is described Non- characteristic gene sequence includes the nucleic acid sequence from T cell receptor D and/or T cell receptor A, and described with non-characteristic The probe that gene order combines includes sequence selected from the group below: SEQ ID NO.201 is shown, SEQ ID NO.202 is shown, SEQ Shown in ID NO.203, shown in SEQ ID NO.204, shown in SEQ ID NO.205 and sequence shown in SEQ ID NO.206.Institute Stating non-characteristic gene sequence includes the nucleic acid sequence from T cell receptor B, and described can expand non-characteristic gene sequence Reagent or device include sequence selected from the group below: shown in SEQ ID NO.158, shown in SEQ ID NO.159, SEQ ID Shown in NO.160, SEQ ID NO.161 is shown, SEQ ID NO.162 is shown, SEQ ID NO.163 is shown, SEQ ID Shown in NO.164, SEQ ID NO.165 is shown, SEQ ID NO.166 is shown, SEQ ID NO.167 is shown, SEQ ID Shown in NO.168, shown in SEQ ID NO.169 and sequence shown in SEQ ID NO.170.The non-characteristic gene sequence packet The nucleic acid sequence from T cell receptor B is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below Column: SEQ ID NO.207 is shown, SEQ ID NO.208 is shown, SEQ ID NO.209 is shown, SEQ ID NO.210 is shown, Shown in SEQ ID NO.211, SEQ ID NO.212 is shown, SEQ ID NO.213 is shown, SEQ ID NO.214 is shown, SEQ Shown in ID NO.215, SEQ ID NO.216 is shown, SEQ ID NO.217 is shown, SEQ ID NO.218 is shown and SEQ ID Sequence shown in NO.219.The non-characteristic gene sequence includes the nucleic acid sequence from Tal 1, and it is described can expand it is non- The reagent or device of characteristic gene sequence include sequence selected from the group below: shown in SEQ ID NO.171 and SEQ ID NO.172 Shown in sequence.The non-characteristic gene sequence includes the nucleic acid sequence from Tal 1, and described with non-characteristic gene sequence The probe that column combine includes sequence selected from the group below: shown in SEQ ID NO.220 and sequence shown in SEQ ID NO.221.

In the recognition unit, for characteristic gene sequence described in specific amplification reagent (such as comprising one Group or multiple groups can carry out the primer of specific nucleic acid amplification for the characteristic gene sequence) nucleic acid amplification reaction can be passed through (for example, qPCR reacts) is expanded, to be specific to the characteristic gene sequence of the subject leukaemia cell described in obtaining.

The recognition unit is specific to the subject leukaemia cell's described in can expanding by carrying out qPCR reaction Characteristic gene sequence.The recognition unit may include material and reagent needed for carrying out qPCR reaction.For example, the identification is single Member includesGreen I, archaeal dna polymerase, dNTP, amplification buffer and one or more groups of it can effectively obtain (example Such as, expand) the primer combination of the characteristic gene sequence for being specific to the subject leukaemia cell.The DNA polymerization Enzyme can be Taq DNA polymerase.The amplification buffer can be MasterMIX.

When carrying out qPCR reaction in the recognition unit, the system of the qPCR reaction can be single with the identification Difference of the member containing component and adjust accordingly, described be specific to the subject as long as effectively can specifically expand The characteristic gene sequence of leukaemia cell.For example, in terms of the system of 25 μ L, the system of the qPCR reaction can be with are as follows: 2 μ L DNA profiling, 0.5 μ L forward primer, 0.5 μ L reverse primer, 12.5 μ LPremixEx Taq ^TMWith 9.5 μ L dH₂O.In some embodiments, in terms of the system of 20 μ L, the system of qPCR reaction can be with are as follows: 2 μ L DNA profilings, 0.3 μ L forward primer, 0.3 μ L reverse primer, 0.4 μ L fluorescence probe, 10 μ L Taqman Universal PCR Master Mix and 7μL dH₂O。

When carrying out qPCR reaction in the recognition unit, the condition of the qPCR reaction can be specific to institute with described It states the length difference of the characteristic gene sequence of subject leukaemia cell and adjusts accordingly, as long as can be effectively special Property expand the characteristic gene sequence for being specific to the subject leukaemia cell.The condition of the qPCR reaction It can be with are as follows: 95 DEG C recycle 40 times, 95 DEG C for 30 seconds, 94 DEG C for 5 seconds and 60 DEG C for 34 seconds and kept for 4 DEG C after 1 minute in 15 seconds, 60 DEG C.Alternatively, can With are as follows: 94 DEG C are after ten minutes, and 94 DEG C recycle 35 times, 72 DEG C for 45 seconds in 45 seconds and 72 DEG C in 45 seconds, 55 DEG C and keep 4 DEG C after 15 minutes.Institute Stating recognition unit may include the instrument and equipment for carrying out the qPCR reaction.For example, ABI7000/7700/7900HT、 LightCycler Real Time PCR or Smart Cycler II System Real Time PCR amplification instrument.

In this application, term " probe " typically refers to the sequence specific with optical activation when hybridizing with amplified production Property oligonucleotide probe.The probe can be connected to any optical activity report agent (for example, dyestuff), and may also include energy Enough block optically active quencher of associated dyestuff.Can include with the non-limiting example of the probe for agent of giving a report TaqMan probe, TaqMan Tamara probe, TaqMan MGB probe or Lion probe.The optical activity reports the non-of agent Limitative examples include SYBR green, and SYBR is blue, DAPI, propidium iodide (propidium iodine), Hoeste, SYBR gold, bromine Change second ingot, acridine, proflavin, acridine orange, acridine yellow, fluorescent coumarin (fluorcoumanin), ellipticine, daunomycin, Chloroquine, distamycin D, chromomycin, Homidium Bromide (homidium), mithramycin, more pyridine ruthenium (ruthenium Polypyridyl), Anthramycin (anthramycin), phenanthridines and acridine, ethidium bromide, propidium iodide, the own pyridine of iodate (hexidium iodide), dihydro second ingot, second ingot homodimer -1 and second ingot homodimer -2, single Azide second ingot (ethidium monoazide) and ACMA, Hoechst 33258, Hoechst 33342, Hoechst 34580, DAPI, a word used for translation Pyridine orange, 7-AAD, actinomycin D, LDS751, hydroxystilbamidine (hydroxystilbamidine), SYTOX is blue, and SYTOX is green, SYTOX orange, POPO-1, POPO-3, YOYO-1, YOYO-3, TOTO-1, TOTO-3, JOJO-1, LOLO-1, BOBO-1, BOBO- 3, PO-PRO-1, PO-PRO-3, BO-PRO-1, BO-PRO-3, TO-PRO-1, TO-PRO-3, TO-PRO-5, JO-PRO-1, LO- PRO-1, YO-PRO-1, YO-PRO-3, PicoGreen, OliGreen, RiboGreen, SYBR gold, the SYBR green II of green I, SYBR, SYBR DX, SYTO-40, -41, -42, -43, -44, -45 (indigo plant), SYTO-13, -16, -24, -21, -23, -12, -11, -20, - 22, -15, -14, -25 (green), SYTO-81, -80, -82, -83, -84, -85 (orange), SYTO-64, -17, -59, -61, -62, - 60, -63 (red), fluorescein, fluorescein isothiocynate (FITC), tetramethylrhodamine isothiocyanates (TRITC), rhodamine, Tetramethylrhodamine, R-PE, Cy-2, Cy-3, Cy-3.5, Cy-5, Cy5.5, Cy-7, texas Red (Texas Red), Phar-Red, allophycocyanin (APC), Sybr green I, Sybr green II, Sybr gold, CellTracker is green, 7-AAD, second Ingot homodimer I, second ingot homodimer II, second ingot homodimer III, ethidium bromide, umbelliferone, eosin, green are glimmering Photoprotein, erythrosine, cumarin, methylcoumarin, pyrene, peacock green, Stilbene, fluorescein, cascade blue (cascade blue), dichloro Atrazin fluorescein, dansyl Cl, fluorescence Lanthanide Complexes (such as those include the complex compound of europium and terbium), carboxyl tetrachloro fluorescence Element, 5 and/or 6- Fluoresceincarboxylic acid (FAM), 5- (or 6-) iodacetyl amido fluorescein, 5- { [2 (and 3) -5- (acetyl group mercaptos Base)-succinyl group] amino } fluorescein (SAMSA- fluorescein), Sulforhodamine B sulfonic acid chloride, 5 and/or 6 carboxyrhodamines (ROX), 7- amino-methyl-cumarin, 7- amino -4- methylcoumarin -3- acetic acid (AMCA), BODIPY fluorogen, 8- methoxy Base pyrene -1,3,6- trisulfonic acid trisodium salt, 3,6- disulfonic acid -4- amino-naphthalimide, phycobniliprotein, AlexaFluor 350,405,430,488,532,546,555,568,594,610,633,635,647,660,680,700,750 and 790 dyestuff, The dyestuff of DyLight350,405,488,550,594,633,650,680,755 and 800 or other fluorogens.

System described herein, it may include 2 or 2 or more detection modules, wherein at least one detection module are used In the presence and/or ratio that detect wherein leukaemia cell before the subject receives treatment, and wherein at least one is examined Survey presence and/or ratio that module is used to detect wherein leukaemia cell after the subject receives the treatment.

In this application, term " treatment " typically refers to drug therapy and/or radiotherapy.The drug can be selected from albumen Kinase inhibitor, apoptosis regulators, rush gene splitting drug, cell division inhibitor, MDM2 class oncogene inhibitor, Antibody class drug and/or other chemotherapeutics additives.The drug may also include one of the following group or a variety of: Changchun is new Alkali, dexamethasone, cyclophosphamide, vincristine, dexamethasone and leunase combination, NSC750854, topology are replaced Health, Volasertib, Serdemetan, JNJ-26481585 and KTP-330.The kinases inhibitor may include AZD8055, MLN0128, GSK690693, MK-2206, SAR245408, rapamycin, MLN0128, selumetinib, AZD6244, PCI-32765, ibrutinib, SGI-1776, dinaciclib, VS-4718, temsirolimus, Sorafenib, sunitinib, ruxolitinib, AZD1480 and/or dasatinib.The apoptosis regulators can wrap It includes ABT-263 (navitoclax), ABT-199 (venetoclax), LCL161 and/or birinapant.The MDM2 class causes Oncogene inhibitor may include MK-8242 (SCH 900242) and/or RG7112.The rush gene splitting drug may include PR- 104, cytarabine (CPX-351), daunorubicin (Vyxeos), topotecan, clofarabine and/or temozolomide.The cell division inhibitor may include eribulin, ispinesib, bortezomib, Carfilzomib and/or alisertib.The antibody class drug may include SAR3419, blinatumomab, and/or inotuzumab.Other described chemotherapeutics additives may include alvespimycin, AT13387, PF-03084014, RO- 4929097, MLN4924, PG11047, CX-5461, BMN-673, selinexor, curaxin and/or CBL0137.It is described to put Penetrate the irradiation that treatment may include x-ray, alpha ray, β ray, gamma-rays, proton beam or other particles beams.

In some embodiments, before applying the treatment to subject, the sample for being originated from the subject is obtained (for example, blood cell samples, or the DNA sample extracted from the cell), and the sample is serially diluted (for example, dilute Release 10 times, 100 times, 1000 times, 10,000 times, 100,000 times or more).Then, using the identification module respectively to described through dilute The characteristic gene sequence of subject is expanded (for example, qPCR expand) in the sample released, thus establish it is a series of right It should be in the standard amplification curve of various concentration (for example, different DNA concentrations) sample.Applying the treatment and then secondary acquisition The sample of the subject by treatment (for example, corresponding blood cell samples, or extracts corresponding from the cell DNA sample), and expanded using the characteristic gene sequence of the identification module to subject in the sample (for example, Identical amplification method, such as same qPCR amplification method when with the production standard amplification curve).

One can be spaced between the amplification before applying the treatment and the amplification after the application treatment The section time.For example, interval at least 1 day, at least 2 days, at least 3 days, at least 4 days, at least 5 days, at least 6 days, at least 7 days, at least 8 It, at least 9 days, at least 10 days, at least 11 days, at least 12 days.Then, can by will apply obtain after the treatment for described Identification and/or amplification (for example, the qPCR is expanded) result of the characteristic gene sequence of subject and the standard amplification are bent Line is compared, to judge the ratio of the characteristic gene sequence of leukaemia cell in the subject after the treatment.

In this application, term " treatment prompt unit " is typically referred to according to leukaemia cell in subject detected In the presence of and/or the ratio prompt functional unit of successive treatment scheme that corresponding subject is used.For example, the treatment prompt is single Member may include the presence of leukaemia cell and/or the detecting element (for example, flow cytometer) of ratio and right in detection subject The decision element that the result of presence and/or ratio that the detecting element detects is judged is (for example, CPU processor, operation Program).The treatment prompt unit may also include the result of judgement for showing the decision element prompt element (for example, Display, display screen, instantaneous speech power etc.).

The detecting element detects the presence and/or ratio of leukaemia cell in subject detected, and will test Presence and/or the result of ratio be transferred to the decision element.The decision element according to its setting, according in subject The ratio of leukaemia cell uses the condition of different successive treatment schemes, to the result of presence and/or ratio that transmission obtains Judged, obtain selection successive treatment scheme as a result, and the result can be transferred to the prompt element.The prompt member The level results that transmission obtains are fed back the drug candidate out by forms such as image, sound and are applicable in the subject by part Situation, so that the selection to subsequent clinical treatment provides reliable reference.

For example, can be prompted according to the size of the ratio of leukaemia cell in subject detected to corresponding subject Using different successive treatment schemes.For example, when ratio < 1/10 of leukaemia cell in subject detected⁴When, it can be shown that The treatment is successful, it is no longer necessary to carry out additional treatment；When ratio > 1/ of leukaemia cell in subject detected 10⁴When, it can be shown that the treatment is incomplete, it is also necessary to carry out the treatment of a new round or improve the intensity for the treatment of.

Ratio > 1/10 of leukaemia cell in subject detected⁴In the case where, it can further analyze detected The size of the ratio of leukaemia cell in subject, to judge whether to be suitable for carrying out bone-marrow transplantation to the subject.For example, If 1/10⁴Ratio < 1/10 of leukaemia cell in <subject detected³, can be shown that can carry out marrow to the subject Transfer operation；If ratio > 1/10 of leukaemia cell in subject detected³, can be shown that in subject's body and exist greatly The leukaemia cell of amount, since a possibility that higher leukaemia cell's residual degree can make leukemia relapse, greatly increases, because This is considered as the subject in this case and is unsuitable for carrying out bone marrow transplant, and should receive other treatment or improve treatment Intensity.

In this application, after the disease characterization that term " recurrence " is often referred to patient is eliminated or mitigates, disease is characterized once again The phenomenon that occurring or aggravating.

In this application, term " other treatment " is typically referred in addition to subject detected is it has been accepted that therapeutic modality Treatment method in addition.

A specific embodiment of the described method and system of the application is shown in Fig. 6.Wherein in step or module In " 1 " (i.e. amplification unit) to target gene (for example, target gene may include IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1 etc., and genetic recombination can wherein occur) expanded；In step or module " 2 " The amplified production obtained by described " 1 " is separated in (i.e. identification unit and analytical unit) and obtained target is expanded Increase segment and carry out nucleic acid sequencing, so that it is determined that being specific to the gene order of the characteristic amplified production of the leukaemia cell of subject； Quantitative analysis is carried out to the gene order obtained by " 2 " in step or module " 3 " (i.e. recognition unit), wherein passing through design tool There are personalized primer pair, the general probe in conjunction with non-characteristic gene sequence-specific, Yi Jite of specific nucleotide sequence The universal primer pair of non-characteristic gene sequence described in specific amplification is thin from subject's leukaemia in sample to quantitatively detect The content of the characteristic amplified production of born of the same parents, and then the residual level of leukaemia cell in subject is detected on a molecular scale.

In the application, the operating process of the leukaemia diagnosis and therapy system may include content below: subject goes to hospital just It is diagnosed as after examining with leukaemia, at this time by the modeling module of leukaemia diagnosis and therapy system described herein, is constructed tested The specific humanized disease model of person；Hereafter subject is subjected to this field routine, standardized treatment (for example, standardization Treat), after the treatment end, then by the detection module of the leukaemia diagnosis and therapy system, subject is detected on a molecular scale The presence and/or ratio of leukaemia cell；If the result of detection illustrates that the subject has biggish probability that can recur leukaemia, Or the ratio of leukaemia cell is excessively high, then illustrates that subject produces drug resistance to above-mentioned conventional, standardized treatment Property；It can be seen that in such cases it need the specific active drug in subject, therefore utilize leukaemia described herein The drug candidate screening module of diagnosis and therapy system (is changed using the specific humanized disease model screening drug candidate of the subject Treat medicine, targeting medicine), according to the level results of previously described drug candidate, choose the drug candidate guidance of class 5 or class 6 Clinical treatment makes the subject receive treatment through screen drug of the specificity in itself；After treating a period of time, if by institute The detection module for stating leukaemia diagnosis and therapy system shows that the ratio that subject leukaemia cell is detected on molecular level is very low or does not have, Then think that leukaemia diagnosis and therapy system screening drug obtained is very significant to the curative effect of the subject.

The application further relates to embodiment in detail below:

1. a kind of system for carrying out leukaemia diagnosis and treatment comprising:

A) modeling module, is used to construct the specific humanized disease model of subject, and the subject is leukaemic；

B) at least one detection module is used to detect the presence and/or ratio of leukaemia cell in the subject；

Wherein the modeling module includes:

A1) sample unit, it includes the leukaemia cell's suspension for being originated from the subject, leukaemia in the suspension The density of cell is 2x10⁷/ ml to 5x10⁷/ml；

A2) radiating element, it includes radioactivity processing units, for carrying out radioactivity to immune-deficient mice appropriate Processing；

A3 it) is inoculated with unit, is used to exempt from leukaemia cell's inoculation of suspension liquid to what is handled through the radiating element In epidemic disease deficient mice；

Wherein at least one described detection module includes:

B1) amplification unit, it includes the reagents for capableing of specific amplification target gene, to obtain target specificity amplified production.

2. further including that c) drug candidate screening module, the drug candidate screen according to system described in embodiment 1 Module includes the first drug candidate group and the second drug candidate group；

Wherein the first drug candidate group includes one or more drugs selected from the group below: vincristine, dexamethasone, Leunase, purine nucleosides analog derivative, proteasome inhibitor, mTOR inhibitors, reaches topoisomerase enzyme inhibitor Sand for Buddhist nun, antibody class drug, all-trans retinoic acid, DNA damage inducer, folic acid reductase inhibitor, deacetylate inhibitor, Polyceptor tyrosine kinase inhibitor, cytotoxic drug and retinamide；And

Wherein the second drug candidate group includes one or more drugs selected from the group below: kinases inhibitor, thin Born of the same parents' apoptosis regulator, DNA distintegrant, cell division inhibitor, MDM2 class oncogene inhibitor, antibody class drug and other changes Treat pharmaceutical addition agent.

3. the system according to any one of embodiment 1-2, wherein the modeling module further includes a4) separation list Member, the separative unit includes monocyte separation medium, for being centrifugated the leukaemia cell of the subject.

4. the system according to any one of embodiment 1-3, wherein the modeling module further includes a5) T cell goes Except unit, it is used to remove the T cell being originated from the sample of non-T cell type leukaemia subject, to obtain the leukaemia Cell suspending liquid.

5. the system according to any one of embodiment 3-4, wherein the monocyte separation medium is Ficoll, and The centrifuge separation is that density gradient centrifugation separates.

6. the system according to any one of embodiment 1-5, wherein including in leukaemia cell's suspension RPMI cell culture medium.

7. the system according to any one of embodiment 4-6, wherein the T cell being removed is CD3 positive T cell.

8. according to system described in embodiment 7, wherein the T cell removal unit contains the magnetic bead comprising CD3 antibody.

9. the system according to any one of embodiment 7-8, wherein the T cell removal unit also includes highfield.

10. the system according to any one of embodiment 1-9, wherein the leukaemia is that acute lymphocytic is white Blood disease, and the immune-deficient mice is NSG mouse.

11. the system according to any one of embodiment 1-9, wherein the leukaemia is acute myeloid leukaemia, And the immune-deficient mice is MISTRG mouse.

12. according to system described in embodiment 10, wherein the radioactivity processing unit is X-ray emitter, and Its dose of radiation generated is 200cGy to 300cGy.

13. according to system described in embodiment 11, wherein the radioactivity processing unit is X-ray emitter, and Its dose of radiation generated is 550cGy to 650cGy.

14. the system according to any one of embodiment 1-13, wherein the radiating element is arranged to predetermined Inoculation time before 12-24 hour in the radioactivity processing is carried out to the immune-deficient mice.

15. the system according to any one of embodiment 1-14, wherein the age of the immune-deficient mice is 5-7 weeks.

16. the system according to any one of embodiment 1-15, wherein the modeling module further includes a6) infrared photograph It penetrates and heating unit, for carrying out infrared light irradiation and heating to the immune-deficient mice.

17. the system according to any one of embodiment 1-16, wherein the modeling module further includes a7) monitoring unit, For monitoring the leukaemia cell from the subject in the intracorporal growing state of the immune-deficient mice through being inoculated with.

18. according to system described in embodiment 17, wherein the monitoring unit includes detecting in the mouse peripheral blood The reagent and device of the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell.

19. according to system described in embodiment 18, wherein the modeling module further includes a8) spleen processing unit, it should Spleen processing unit includes resolution element, broken component and levitated element, as huCD45 in the mouse peripheral blood through being inoculated with +, the ratio of huCD19+, huCD3+, and/or huCD33+ cell is when being at least 50%, the resolution element can be passed through and separate institute The spleen for stating mouse is crushed the spleen of the separated mouse by the broken component to obtain spleen cell, and passes through The spleen cell is prepared as leukaemia cell's suspension by the levitated element.

20. the system according to any one of embodiment 1-19, wherein the modeling module further includes a9) liquid nitrogen, For leukaemia cell's suspension described in stored frozen.

21. the system according to any one of embodiment 2-20, wherein the drug candidate screening module further includes Monitoring unit, it is intracorporal in the immune-deficient mice through being inoculated with for monitoring the leukaemia cell from the subject Growing state.

22. according to system described in embodiment 21, wherein the monitoring unit includes detecting in the mouse peripheral blood The reagent and device of the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell, and the monitoring unit is set It is set to, when the ratio reaches 1%-5%, prompt can apply drug candidate to the mouse.

23. the system according to embodiment 21 or 22, wherein the monitoring unit includes detecting the mouse periphery The reagent and device of the ratio of huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in blood, and the monitoring unit It is arranged to, after medicament administration, continues to monitor huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in institute The variation of proportion in mouse peripheral blood cell is stated, and prompts the effect of the drug candidate according to the variation.

24. according to system described in embodiment 23, wherein the monitoring unit is arranged to: when the application candidate medicine After object, monitor huCD45+, huCD19+, huCD3+, and/or huCD33+ cell in the mouse peripheral blood cell When proportion keeps below 1% and maintains the ratio at least 2 weeks, then it is effective for prompting the drug candidate.

25. the system according to any one of embodiment 2-24, wherein the first drug candidate group includes being selected from One or more drugs of the following group: vincristine, dexamethasone, leunase, topotecan, clofarabine, card are non- Help rice, tesirolimus, Dasatinib, bortezomib, CD19 antibody, all-trans retinoic acid, adriamycin, ninopterin, SAHA, Sutent, cyclophosphamide and retinamide.

26. the system according to any one of embodiment 2-25, wherein described in the second drug candidate group Kinases inhibitor includes one or more drugs selected from the group below: AZD8055, MLN0128, GSK690693, MK-2206, SAR245408、rapamycin、MLN0128、selumetinib、AZD6244、PCI-32765、ibrutinib、SGI-1776、 Dinaciclib, VS-4718, sorafenib, sunitinib and AZD1480.

27. the system according to any one of embodiment 2-26, wherein described in the second drug candidate group Apoptosis regulators include one or more drugs selected from the group below: ABT-263 (navitoclax), ABT-199 (venetoclax), LCL161 and birinapant.

28. the system according to any one of embodiment 2-27, wherein described in the second drug candidate group DNA distintegrant includes one or more drugs selected from the group below: PR-104, cytarabine (CPX-351), daunorubicin (Vyxeos) and temozolomide.

29. the system according to any one of embodiment 2-28, wherein described in the second drug candidate group Cell division inhibitor includes one or more drugs selected from the group below: eribulin, ispinesib and alisertib.

30. the system according to any one of embodiment 2-29, wherein described in the second drug candidate group MDM2 class oncogene inhibitor includes one or more drugs selected from the group below: MK-8242 (SCH 900242) and RG7112.

31. the system according to any one of embodiment 2-30, wherein described in the second drug candidate group Antibody class drug includes one or more drugs selected from the group below: SAR3419.

32. the system according to any one of embodiment 2-31, wherein described in the second drug candidate group Other chemotherapeutics additives include one or more drugs selected from the group below: alvespimycin, AT13387, PF- 03084014, RO-4929097, MLN4924, PG11047, CX-5461, BMN-673, selinexor, bortezomib, Curaxin and CBL0137.

33. the system according to any one of embodiment 2-32, wherein including length in the first drug candidate group Spring new alkali, and the vincristine is suitable for being applied to the specific humanized disease model of the subject according to following requirement: it is suitable In by intraperitoneal injection application, each amount of application is 0.5mg/kg, and application is primary weekly and persistently applies 3-6 weeks.

34. the system according to any one of embodiment 2-33, wherein including ammonia in the first drug candidate group First folic acid, and the ninopterin is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 3-6mg/kg, and application is primary every two weeks, and persistently applies 6-10 weeks.

35. the system according to any one of embodiment 2-34, wherein including ground in the first drug candidate group Sai meter Song, and the dexamethasone is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 10-20mg/kg, once-a-day administration and persistently application 3-6 weeks.

36. the system according to any one of embodiment 2-35, wherein include in the first drug candidate group Ah Mycin, and the adriamycin is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through vein Injection application, each amount of application are 0.5-3mg/kg, and application is primary weekly and persistently applies 3-6 weeks.

37. the system according to any one of embodiment 2-36, wherein including a left side in the first drug candidate group Asparaginase is revolved, and the leunase is suitable for being applied to the leukaemia humanization disease mould according to following requirement Type: suitable for by intraperitoneal injection application, each amount of application is 500-2000KU/kg, once-a-day administration and persistently application 3-6 weeks.

38. the system according to any one of embodiment 2-37, wherein including topology in the first drug candidate group For health, and the topotecan is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through abdominal cavity Injection application, each amount of application are 0.1-5mg/kg, once-a-day administration and are persistently applied 2-8 weeks, are discontinued one week after two weeks.

39. the system according to any one of embodiment 2-38, wherein including chlorine in the first drug candidate group Farad shore, and the clofarabine is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 10-100mg/kg, once-a-day administration and persistently application 3-6 weeks.

40. the system according to any one of embodiment 2-39, wherein including card in the first drug candidate group Fei Zuo meter, and the Carfilzomib is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intravenous injection application, each amount of application are 0.1-10mg/kg, apply weekly secondary and persistently apply 3-6 weeks.

41. the system according to any one of embodiment 2-40, wherein including replacing in the first drug candidate group Sirolimus, and the tesirolimus is suitable for being applied to the leukaemia humanization disease model according to following requirement: it is suitable for It is applied by intraperitoneal injection, each amount of application is 5-50mg/kg, once-a-day administration and persistently application 1-3 weeks.

42. the system according to any one of embodiment 2-41, wherein including reaching in the first drug candidate group Sand replaces Buddhist nun, and the Dasatinib is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through It is administered orally, each amount of application is 5-50mg/kg, once-a-day administration and persistently application 2-6 weeks.

43. the system according to any one of embodiment 2-42, wherein including boron in the first drug candidate group Bortezomib, and the bortezomib is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 0.1-20mg/kg, apply weekly secondary and persistently apply 4-8 weeks.

44. the system according to any one of embodiment 2-43, wherein including in the first drug candidate group SAR3419, and the SAR3419 is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 0.5-50mg/kg, and application is primary weekly and persistently applies 4-8 weeks.

45. the system according to any one of embodiment 2-44, wherein including complete in the first drug candidate group Retinotic acid, and the all-trans retinoic acid is suitable for being applied to the leukaemia humanization disease model according to following requirement: Suitable for being applied by intraperitoneal injection, each amount of application is 0.5-10mg/kg, and application is primary weekly and persistently applies 2-6 weeks.

46. the system according to any one of embodiment 2-45, wherein including in the first drug candidate group SAHA, and the SAHA is suitable for being applied to the leukaemia humanization disease model according to following requirement: it is suitable for infusing by abdominal cavity Application is penetrated, each amount of application is 100-500mg/kg, once-a-day administration and persistently application 3-6 weeks.

47. the system according to any one of embodiment 2-46, wherein including relaxing in the first drug candidate group Buddhist nun replaces Buddhist nun, and the Sutent is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through It is administered orally, each amount of application is 10-100mg/kg, once-a-day administration and persistently application 2-6 weeks.

48. the system according to any one of embodiment 2-47, wherein including ring in the first drug candidate group Phosphamide, and the cyclophosphamide is suitable for being applied to the leukaemia humanization disease model according to following requirement: suitable for passing through Intraperitoneal injection application, each amount of application are 30-300mg/kg, once-a-day administration and persistently application 2-6 weeks.

49. the system according to any one of embodiment 2-48, wherein including dimension in the first drug candidate group Formyl phenol amine, and the retinamide is suitable for being applied to the leukaemia humanization disease model according to following requirement: it is suitable for It is applied by intraperitoneal injection, each amount of application is 20-200mg/kg, once-a-day administration and persistently application 2-6 weeks.

50. the system according to any one of embodiment 1-49, wherein the leukaemia includes B cell acute lymphoblastic Chronic myeloid leukemia and/or T cell acute lymphoblastic leukemia.

51. the system according to any one of embodiment 1-50, wherein the target gene includes selected from the group below One or more genes, its segment or its variant: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell Receptor G and Tal 1.

52. according to system described in embodiment 51, wherein the target gene includes IgH gene rearrangement, and the energy The reagent of enough specific amplification target genes includes combining selected from following one or more groups of primers:

1) forward primer: VH1/7, reverse primer: JH cons；

2) forward primer: VH2, reverse primer: JH cons；

3) forward primer: VH3, reverse primer: JH cons；

4) forward primer: VH4, reverse primer: JH cons；

5) forward primer: VH5, reverse primer: JH cons；

6) forward primer: VH6, reverse primer: JH cons；

7) forward primer: DH1, DH4, DH5 and DH7, reverse primer: JH cons；

8) forward primer: DH2, reverse primer: JH cons；

9) forward primer: DH3, reverse primer: JH cons；With

10) forward primer: DH6, reverse primer: JH cons.

53. according to system described in embodiment 52, wherein the primer JH cons includes sequence selected from the group below: SEQ Sequence shown in ID NO.19；With sequence shown in SEQ ID NO.20.

54. the system according to any one of embodiment 52-53, wherein the primer VH1/7 includes to be selected from the group Sequence: shown in SEQ ID NO.1, shown in SEQ ID NO.2 and sequence shown in SEQ ID NO.3.

55. the system according to any one of embodiment 52-54, wherein the primer VH2 includes selected from the group below Sequence: shown in SEQ ID NO.4, shown in SEQ ID NO.5 and sequence shown in SEQ ID NO.6.

56. the system according to any one of embodiment 52-55, wherein the primer VH3 includes selected from the group below Sequence: shown in SEQ ID NO.7, shown in SEQ ID NO.8 and sequence shown in SEQ ID NO.9.

57. the system according to any one of embodiment 52-56, wherein the primer VH4 includes selected from the group below Sequence: shown in SEQ ID NO.10, shown in SEQ ID NO.11 and sequence shown in SEQ ID NO.12.

58. the system according to any one of embodiment 52-57, wherein the primer VH5 includes selected from the group below Sequence: shown in SEQ ID NO.13, shown in SEQ ID NO.14 and sequence shown in SEQ ID NO.15.

59. the system according to any one of embodiment 52-58, wherein the primer VH6 includes selected from the group below Sequence: shown in SEQ ID NO.16, SEQ ID NO.17 and sequence shown in SEQ ID NO.18.

60. the system according to any one of embodiment 52-59, wherein the primer DH1 includes selected from the group below Sequence: shown in SEQ ID NO.21 and sequence shown in SEQ ID NO.22.

61. the system according to any one of embodiment 52-60, wherein the primer DH2 includes selected from the group below Sequence: shown in SEQ ID NO.23 and sequence shown in SEQ ID NO.24.

62. the system according to any one of embodiment 52-61, wherein the primer DH3 includes selected from the group below Sequence: shown in SEQ ID NO.25 and sequence shown in SEQ ID NO.26.

63. the system according to any one of embodiment 52-62, wherein the primer DH4 includes selected from the group below Sequence: shown in SEQ ID NO.27 and sequence shown in SEQ ID NO.28.

64. the system according to any one of embodiment 52-63, wherein the primer DH5 includes selected from the group below Sequence: shown in SEQ ID NO.29 and sequence shown in SEQ ID NO.30.

65. the system according to any one of embodiment 52-64, wherein the primer DH6 includes selected from the group below Sequence: shown in SEQ ID NO.31 and sequence shown in SEQ ID NO.32.

66. the system according to any one of embodiment 52-65, wherein the primer DH7 includes selected from the group below Sequence: shown in SEQ ID NO.33 and sequence shown in SEQ ID NO.34.

67. the system according to any one of embodiment 51-66, wherein the target gene includes IgK gene weight Row, and the reagent for capableing of specific amplification target gene includes combining selected from following one or more groups of primers:

1) forward primer: Vk1, reverse primer: Kdel；

2) forward primer: Vk2, reverse primer: Kdel；

3) forward primer: Vk3, reverse primer: Kdel；With

4) forward primer: Intron RSS, reverse primer: Kdel.

68. according to system described in embodiment 67, wherein the primer Vk1 includes sequence selected from the group below: SEQ ID Shown in NO.35 and sequence shown in SEQ ID NO.36.

69. the system according to any one of embodiment 67-68, wherein the primer Vk2 includes selected from the group below Sequence: shown in SEQ ID NO.37 and sequence shown in SEQ ID NO.38.

70. the system according to any one of embodiment 67-69, wherein the primer Vk3 includes selected from the group below Sequence: shown in SEQ ID NO.39 and sequence shown in SEQ ID NO.40.

71. the system according to any one of embodiment 67-70, wherein the primer Vk4 includes selected from the group below Sequence: shown in SEQ ID NO.41 and sequence shown in SEQ ID NO.42.

72. the system according to any one of embodiment 67-71, wherein the primer I ntron RSS includes to be selected from The sequence of the following group: shown in SEQ ID NO.43 and sequence shown in SEQ ID NO.44.

73. the system according to any one of embodiment 67-72, wherein the primer Kdel includes selected from the group below Sequence: shown in SEQ ID NO.45 and sequence shown in SEQ ID NO.46.

74. the system according to any one of embodiment 51-73, wherein the leukaemia includes the acute leaching of B cell Bar chronic myeloid leukemia, the target gene includes the gene rearrangement in T cell receptor region；The gene in the T cell receptor region Including one or more selected from the group below: T cell receptor A, T cell receptor D, T cell receptor B and T cell receptor G；And it is described The reagent for capableing of specific amplification target gene includes combining selected from following one or more groups of primers:

1) forward primer: Vd2, reverse primer: Dd3；

2) forward primer: Dd2, reverse primer: Dd3；

3) forward primer: Vd2, reverse primer: Ja29；

4) forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；

5) forward primer: Vg1, reverse primer: Jg1 and Jg2；

6) forward primer: Vg2, reverse primer: Jg1 and Jg2；

7) forward primer: Vg4, reverse primer: Jg1 and Jg2；

8) the more primers of TCRB combine A；

9) the more primers of TCRB combine B；With

10) the more primers of TCRB combine C.

75. according to system described in embodiment 74, wherein the primer Vg1 includes sequence selected from the group below: SEQ ID Shown in NO.47 and sequence shown in SEQ ID NO.48.

76. the system according to any one of embodiment 74-75, wherein the primer Vg2 includes selected from the group below Sequence: shown in SEQ ID NO.49 and sequence shown in SEQ ID NO.50.

77. the system according to any one of embodiment 74-76, wherein the primer Vg4 includes selected from the group below Sequence: shown in SEQ ID NO.53 and sequence shown in SEQ ID NO.54.

78. the system according to any one of embodiment 74-77, wherein the primer Jg1 includes selected from the group below Sequence: shown in SEQ ID NO.55 and sequence shown in SEQ ID NO.56.

79. the system according to any one of embodiment 74-78, wherein the primer Jg2 includes selected from the group below Sequence: shown in SEQ ID NO.57 and sequence shown in SEQ ID NO.58.

80. the system according to any one of embodiment 74-79, wherein the primer Vd2 includes selected from the group below Sequence: shown in SEQ ID NO.61 and sequence shown in SEQ ID NO.62.

81. the system according to any one of embodiment 74-80, wherein the primer Dd2 includes selected from the group below Sequence: shown in SEQ ID NO.65 and sequence shown in SEQ ID NO.66.

82. the system according to any one of embodiment 74-81, wherein the primer Dd3 includes selected from the group below Sequence: shown in SEQ ID NO.67 and sequence shown in SEQ ID NO.68.

83. the system according to any one of embodiment 74-82, wherein the primer Ja9 includes selected from the group below Sequence: sequence shown in SEQ ID NO.71.

84. the system according to any one of embodiment 74-83, wherein the primer Ja29 includes selected from the group below Sequence: sequence shown in SEQ ID NO.72.

85. the system according to any one of embodiment 74-84, wherein the primer Ja30 includes selected from the group below Sequence: sequence shown in SEQ ID NO.73.

86. the system according to any one of embodiment 74-85, wherein the primer Ja48 includes selected from the group below Sequence: sequence shown in SEQ ID NO.74.

87. the system according to any one of embodiment 74-86, wherein the primer Ja49 includes selected from the group below Sequence: sequence shown in SEQ ID NO.75.

88. the system according to any one of embodiment 74-87, wherein the primer Ja52 includes selected from the group below Sequence: sequence shown in SEQ ID NO.76.

89. the system according to any one of embodiment 74-88, wherein the primer Ja54 includes selected from the group below Sequence: sequence shown in SEQ ID NO.77.

90. the system according to any one of embodiment 74-89, wherein the primer Ja55 includes selected from the group below Sequence: sequence shown in SEQ ID NO.78.

91. the system according to any one of embodiment 74-90, wherein the primer Ja56 includes selected from the group below Sequence: sequence shown in SEQ ID NO.79.

92. the system according to any one of embodiment 74-91, wherein the primer Ja57 includes selected from the group below Sequence: sequence shown in SEQ ID NO.80.

93. the system according to any one of embodiment 74-92, wherein the primer Ja58 includes selected from the group below Sequence: sequence shown in SEQ ID NO.81.

94. the system according to any one of embodiment 74-93, wherein the primer Ja59 includes selected from the group below Sequence: sequence shown in SEQ ID NO.82.

95. the system according to any one of embodiment 74-94, wherein the primer Ja61 includes selected from the group below Sequence: sequence shown in SEQ ID NO.83.

96. the system according to any one of embodiment 74-95, wherein more primer combination A include to be selected from down One or more sequences of group: shown in SEQ ID NO.86, shown in SEQ ID NO.87, sequence shown in SEQ ID NO.88, Shown in SEQ ID NO.89, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID Shown in NO.93, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 institute Show, SEQ ID NO.98 is shown, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ Shown in ID NO.102, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID NO.105 is shown, SEQ ID Shown in NO.106, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID NO.111 is shown, SEQ ID Shown in NO.112, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ ID Shown in NO.116, shown in SEQ ID NO.118, SEQ ID NO.122 and sequence shown in SEQ ID NO.123.

97. the system according to any one of embodiment 74-96, wherein more primer combination B include to be selected from down One or more sequences of group: shown in SEQ ID NO.86, SEQ ID NO.87 is shown, SEQ ID NO.88 is shown, SEQ ID Shown in NO.89, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 institute Show, SEQ ID NO.94 is shown, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ ID Shown in NO.98, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ ID NO.102 Shown in shown, SEQ ID NO.103, shown in SEQ ID NO.104, shown in SEQ ID NO.105, shown in SEQ ID NO.106, Shown in SEQ ID NO.107, SEQ ID NO.108 is shown, SEQ ID NO.117 is shown, SEQ ID NO.119 is shown, SEQ Shown in ID NO.120 and sequence shown in SEQ ID NO.121.

98. the system according to any one of embodiment 74-97, wherein more primer combination C include to be selected from down One or more sequences of group: shown in SEQ ID NO.109, SEQ ID NO.110 is shown, SEQ ID NO.111 is shown, SEQ Shown in ID NO.112, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ ID Shown in NO.116, SEQ ID NO.117 is shown, SEQ ID NO.118 is shown, SEQ ID NO.119 is shown, SEQ ID Shown in NO.120, shown in SEQ ID NO.121, shown in SEQ ID NO.122 and sequence shown in SEQ ID NO.123.

99. according to system described in embodiment 51, wherein the leukaemia includes the white blood of T cell acute lymphoblastic Disease, the target gene include the gene rearrangement in T cell receptor region；The gene in the T cell receptor region includes being selected from down The one or more of group: T cell receptor A, T cell receptor D, T cell receptor B and T cell receptor G；And it is described can specificity The reagent of amplification target gene includes combining selected from following one or more groups of primers:

1) forward primer: Vd1, reverse primer: Jd1；

2) forward primer: Vd2, reverse primer: Jd1；

3) forward primer: Vd3, reverse primer: Jd1；

4) forward primer: Vd2, reverse primer: Dd3；

5) forward primer: Dd2, reverse primer: Dd3；

6) forward primer: Dd2, reverse primer: Jd1；

7) forward primer: Vd2, reverse primer: Ja29；

8) forward primer: Vd2, reverse primer: Ja9, Ja29, Ja30, Ja48, Ja49, Ja52, Ja54, Ja55, Ja56, Ja57, Ja58, Ja59 and Ja61；

9) forward primer: Vg1, reverse primer: Jg1 and Jg2；

10) forward primer: Vg2, reverse primer: Jg1 and Jg2；

11) forward primer: Vg3, reverse primer: Jg1 and Jg2；

12) forward primer: Vg4, reverse primer: Jg1 and Jg2；

13) forward primer: Sildb, reverse primer: Taldb1；

14) the more primers of TCRB combine A；

15) the more primers of TCRB combine B；With

16) the more primers of TCRB combine C.

100. according to system described in embodiment 99, wherein the primer Vg3 includes sequence selected from the group below: SEQ ID Shown in NO.51 and sequence shown in SEQ ID NO.52.

101. the system according to any one of embodiment 99-100, wherein the primer Vd3 includes to be selected from the group Sequence: shown in SEQ ID NO.63 and sequence shown in SEQ ID NO.64.

102. the system according to any one of embodiment 99-101, wherein the primer Vg1 includes to be selected from the group Sequence: shown in SEQ ID NO.47 and sequence shown in SEQ ID NO.48.

103. the system according to any one of embodiment 99-102, wherein the primer Vg2 includes to be selected from the group Sequence: shown in SEQ ID NO.49 and sequence shown in SEQ ID NO.50.

104. the system according to any one of embodiment 99-103, wherein the primer Vg4 includes to be selected from the group Sequence: shown in SEQ ID NO.53 and sequence shown in SEQ ID NO.54.

105. the system according to any one of embodiment 99-104, wherein the primer Jg1 includes to be selected from the group Sequence: shown in SEQ ID NO.55 and sequence shown in SEQ ID NO.56.

106. the system according to any one of embodiment 99-105, wherein the primer Jg2 includes to be selected from the group Sequence: shown in SEQ ID NO.57 and sequence shown in SEQ ID NO.58.

107. the system according to any one of embodiment 99-106, wherein the primer Vd2 includes to be selected from the group Sequence: shown in SEQ ID NO.61 and sequence shown in SEQ ID NO.62.

108. the system according to any one of embodiment 99-107, wherein the primer Dd2 includes to be selected from the group Sequence: shown in SEQ ID NO.65 and sequence shown in SEQ ID NO.66.

109. the system according to any one of embodiment 99-108, wherein the primer Dd3 includes to be selected from the group Sequence: shown in SEQ ID NO.67 and sequence shown in SEQ ID NO.68.

110. the system according to any one of embodiment 99-109, wherein the primer Ja9 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.71.

111. the system according to any one of embodiment 99-110, wherein the primer Ja29 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.72.

112. the system according to any one of embodiment 99-111, wherein the primer Ja30 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.73.

113. the system according to any one of embodiment 99-112, wherein the primer Ja48 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.74.

114. the system according to any one of embodiment 99-113, wherein the primer Ja49 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.75.

115. the system according to any one of embodiment 99-114, wherein the primer Ja52 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.76.

116. the system according to any one of embodiment 99-115, wherein the primer Ja54 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.77.

117. the system according to any one of embodiment 99-116, wherein the primer Ja55 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.78.

118. the system according to any one of embodiment 99-117, wherein the primer Ja56 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.79.

119. the system according to any one of embodiment 99-118, wherein the primer Ja57 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.80.

120. the system according to any one of embodiment 99-119, wherein the primer Ja58 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.81.

121. the system according to any one of embodiment 99-120, wherein the primer Ja59 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.82.

122. the system according to any one of embodiment 99-121, wherein the primer Ja61 includes to be selected from the group Sequence: sequence shown in SEQ ID NO.83.

123. the system according to any one of embodiment 99-122, wherein more primer combination A include to be selected from One or more sequences of the following group: shown in SEQ ID NO.86, SEQ ID NO.87 is shown, SEQ ID NO.88 is shown, SEQ Shown in ID NO.89, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 Shown in shown, SEQ ID NO.94, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ Shown in ID NO.98, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ ID Shown in NO.102, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID NO.105 is shown, SEQ ID Shown in NO.106, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID NO.111 is shown, SEQ ID Shown in NO.112, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ ID Shown in NO.116, shown in SEQ ID NO.118, shown in SEQ ID NO.122 and sequence shown in SEQ ID NO.123.

124. the system according to any one of embodiment 99-123, wherein more primer combination B include to be selected from One or more sequences of the following group: shown in SEQ ID NO.86, SEQ ID NO.87 is shown, SEQ ID NO.88 is shown, SEQ Shown in ID NO.89, SEQ ID NO.90 is shown, SEQ ID NO.91 is shown, SEQ ID NO.92 is shown, SEQ ID NO.93 Shown in shown, SEQ ID NO.94, SEQ ID NO.95 is shown, SEQ ID NO.96 is shown, SEQ ID NO.97 is shown, SEQ Shown in ID NO.98, SEQ ID NO.99 is shown, SEQ ID NO.100 is shown, SEQ ID NO.101 is shown, SEQ ID Shown in NO.102, SEQ ID NO.103 is shown, SEQ ID NO.104 is shown, SEQ ID NO.105 is shown, SEQ ID Shown in NO.106, SEQ ID NO.107 is shown, SEQ ID NO.108 is shown, SEQ ID NO.117 is shown, SEQ ID Shown in NO.119, shown in SEQ ID NO.120 and sequence shown in SEQ ID NO.121.

125. the system according to any one of embodiment 99-124, wherein more primer combination C include to be selected from One or more sequences of the following group: SEQ ID NO.109 is shown, SEQ ID NO.110 is shown, SEQ ID NO.111 is shown, Shown in SEQ ID NO.112, SEQ ID NO.113 is shown, SEQ ID NO.114 is shown, SEQ ID NO.115 is shown, SEQ Shown in ID NO.116, SEQ ID NO.117 is shown, SEQ ID NO.118 is shown, SEQ ID NO.119 is shown, SEQ ID Shown in NO.120, shown in SEQ ID NO.121, shown in SEQ ID NO.122 and sequence shown in SEQ ID NO.123.

126. the system according to any one of embodiment 99-125, wherein the primer Vd1 includes to be selected from the group Sequence: shown in SEQ ID NO.59 and sequence shown in SEQ ID NO.60.

127. the system according to any one of embodiment 99-126, wherein the primer Jd1 includes to be selected from the group Sequence: shown in SEQ ID NO.69 and sequence shown in SEQ ID NO.70.

128. the system according to any one of embodiment 99-127, wherein the primer Sildb includes to be selected from down The sequence of group: sequence shown in SEQ ID NO.84.

129. the system according to any one of embodiment 99-128, wherein the primer Taldb1 includes to be selected from down The sequence of group: sequence shown in SEQ ID NO.85.

130. the system according to any one of embodiment 52-98, wherein the leukaemia is B cell acute lymphoblastic Chronic myeloid leukemia.

131. the system according to any one of embodiment 99-129, wherein the leukaemia is the acute leaching of T cell Bar chronic myeloid leukemia.

132. the system according to any one of embodiment 1-131, wherein the detection module further includes that identification is single Member, which includes the reagent and device for identifying the target specificity amplified production, to identify the target Mark the characteristic amplified production that the leukaemia cell of the subject is originated from specific amplification products.

133. according to system described in embodiment 132, wherein the identification unit includes to expand the target specificity Increase production reagent and device needed for object carries out gel electrophoresis separation.

134. the system according to any one of embodiment 132-133, wherein the detection module further includes analysis Unit, the analytical unit include reagent and device for analyzing the characteristic amplified production, thus obtain be specific to it is described The characteristic gene sequence of the leukaemia cell of subject.

135. according to system described in embodiment 134, wherein the analytical unit includes to expand to produce to the characteristic Reagent and device needed for object carries out nucleic acid sequencing and sequence analysis.

136. according to system described in embodiment 135, wherein the analytical unit includes identifying the characteristic amplification Reagent and device needed for conservative nucleic acid sequence and subject's specific nucleic acid sequence in the nucleic acid sequence of product, to obtain It is specific to the characteristic gene sequence of the subject leukaemia cell.

137. the system according to any one of embodiment 135-136, wherein the detection module further includes identification Unit, the recognition unit include the examination for capableing of the characteristic gene sequence of specific recognition and/or the amplification subject Agent and device, to judge the presence and/or ratio of residual leukemic cell in the subject.

138. according to system described in embodiment 137, wherein the recognition unit includes that specific amplification is specific to institute State the reagent or device of the characteristic gene sequence of subject leukaemia cell.

139. according to system described in embodiment 138, wherein the recognition unit also includes and non-characteristic gene sequence Arrange the probe combined and reagent or device that non-characteristic gene sequence can be expanded.

140. according to system described in embodiment 139, wherein the non-characteristic gene sequence includes being originated from one of the following group Or the nucleic acid sequence of several genes: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1.

141. systems according to embodiment 140, wherein the non-characteristic gene sequence includes the core from IgH Acid sequence, and the reagent that can expand non-characteristic gene sequence or device include sequence selected from the group below: SEQ ID Shown in NO.124, SEQ ID NO.125 is shown, SEQ ID NO.126 is shown, SEQ ID NO.127 is shown, SEQ ID Shown in NO.128, shown in SEQ ID NO.129, shown in SEQ ID NO.130 and sequence shown in SEQ ID NO.131.

142. systems according to any one of embodiment 140-141, wherein the non-characteristic gene sequence packet The nucleic acid sequence from IgH is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: SEQ Shown in ID NO.173, SEQ ID NO.174 is shown, SEQ ID NO.175 is shown, SEQ ID NO.176 is shown, SEQ ID Shown in NO.177, shown in SEQ ID NO.178, shown in SEQ ID NO.179 and sequence shown in SEQ ID NO.180.

143. systems according to any one of embodiment 140-142, wherein the non-characteristic gene sequence packet The nucleic acid sequence from IgK is included, and the reagent that can expand non-characteristic gene sequence or device include selected from the group below Sequence: SEQ ID NO.132 is shown, SEQ ID NO.133 is shown, SEQ ID NO.134 is shown, SEQ ID NO.135 is shown, Shown in SEQ ID NO.136, SEQ ID NO.137 is shown, SEQ ID NO.138 is shown, SEQ ID NO.139 is shown, SEQ Shown in ID NO.140 and sequence shown in SEQ ID NO.141.

144. systems according to any one of embodiment 140-143, wherein the non-characteristic gene sequence packet The nucleic acid sequence from IgK is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: SEQ Shown in ID NO.181, SEQ ID NO.182 is shown, SEQ ID NO.183 is shown, SEQ ID NO.184 is shown, SEQ ID Shown in NO.185, SEQ ID NO.186 is shown, SEQ ID NO.187 is shown, SEQ ID NO.188 is shown, SEQ ID Shown in NO.189 and sequence shown in SEQ ID NO.190.

145. systems according to any one of embodiment 140-144, wherein the non-characteristic gene sequence packet The nucleic acid sequence from T cell receptor G is included, and the reagent that can expand non-characteristic gene sequence or device include to be selected from The sequence of the following group: shown in SEQ ID NO.142, SEQ ID NO.143 is shown, SEQ ID NO.144 is shown, SEQ ID Shown in NO.145, SEQ ID NO.146 is shown, SEQ ID NO.147 is shown, SEQ ID NO.148 is shown, SEQ ID Shown in NO.149, shown in SEQ ID NO.150 and sequence shown in SEQ ID NO.151.

146. systems according to any one of embodiment 140-145, wherein the non-characteristic gene sequence packet The nucleic acid sequence from T cell receptor G is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below Column: SEQ ID NO.191 is shown, SEQ ID NO.192 is shown, SEQ ID NO.193 is shown, SEQ ID NO.194 is shown, Shown in SEQ ID NO.195, SEQ ID NO.196 is shown, SEQ ID NO.197 is shown, SEQ ID NO.198 is shown, SEQ Shown in ID NO.199 and sequence shown in SEQ ID NO.200.

147. systems according to any one of embodiment 140-146, wherein the non-characteristic gene sequence packet Include the nucleic acid sequence from T cell receptor D and/or T cell receptor A, and the examination that non-characteristic gene sequence can be expanded Agent or device include sequence selected from the group below: SEQ ID NO.152 is shown, SEQ ID NO.153 is shown, SEQ ID NO.154 Shown in shown, SEQ ID NO.155, shown in SEQ ID NO.156 and sequence shown in SEQ ID NO.157.

148. systems according to any one of embodiment 140-147, wherein the non-characteristic gene sequence packet Include the nucleic acid sequence from T cell receptor D and/or T cell receptor A, and the probe in conjunction with non-characteristic gene sequence Include sequence selected from the group below: shown in SEQ ID NO.201, SEQ ID NO.202 is shown, SEQ ID NO.203 is shown, SEQ Shown in ID NO.204, shown in SEQ ID NO.205 and sequence shown in SEQ ID NO.206.

149. systems according to any one of embodiment 140-148, wherein the non-characteristic gene sequence packet The nucleic acid sequence from T cell receptor B is included, and the reagent that can expand non-characteristic gene sequence or device include to be selected from The sequence of the following group: shown in SEQ ID NO.158, SEQ ID NO.159 is shown, SEQ ID NO.160 is shown, SEQ ID Shown in NO.161, SEQ ID NO.162 is shown, SEQ ID NO.163 is shown, SEQ ID NO.164 is shown, SEQ ID Shown in NO.165, SEQ ID NO.166 is shown, SEQ ID NO.167 is shown, SEQ ID NO.168 is shown, SEQ ID Shown in NO.169 and sequence shown in SEQ ID NO.170.

150. systems according to any one of embodiment 140-149, wherein the non-characteristic gene sequence packet The nucleic acid sequence from T cell receptor B is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below Column: SEQ ID NO.207 is shown, SEQ ID NO.208 is shown, SEQ ID NO.209 is shown, SEQ ID NO.210 is shown, Shown in SEQ ID NO.211, SEQ ID NO.212 is shown, SEQ ID NO.213 is shown, SEQ ID NO.214 is shown, SEQ Shown in ID NO.215, SEQ ID NO.216 is shown, SEQ ID NO.217 is shown, SEQ ID NO.218 is shown and SEQ ID Sequence shown in NO.219.

151. systems according to any one of embodiment 140-150, wherein the non-characteristic gene sequence packet The nucleic acid sequence from Tal 1 is included, and the reagent that can expand non-characteristic gene sequence or device include to be selected from the group Sequence: shown in SEQ ID NO.171 and sequence shown in SEQ ID NO.172.

152. systems according to any one of embodiment 140-151, wherein the non-characteristic gene sequence packet The nucleic acid sequence from Tal 1 is included, and the probe in conjunction with non-characteristic gene sequence includes sequence selected from the group below: Shown in SEQ ID NO.220 and sequence shown in SEQ ID NO.221.

153. systems according to any one of embodiment 1-152 comprising 2 or 2 or more detection modules, Wherein at least one detection module be used for the subject receive treatment before detect the wherein presence of leukaemia cell and/or Ratio, and wherein at least one detection module is used to detect wherein leukaemia cell's after the subject receives the treatment In the presence of and/or ratio.

154. systems according to any one of embodiment 1-153, wherein the detection module includes treatment prompt Unit, after being used according to the presence of leukaemia cell in subject detected and/or ratio prompt to corresponding subject Continuous therapeutic scheme.

155. systems according to embodiment 154, wherein when the ratio of leukaemia cell in subject detected It is 1/10⁴When following, the treatment prompt unit prompt is described to be treated successfully and without carrying out additional treatment.

156. systems according to any one of embodiment 154-155, wherein when blood white in subject detected The ratio of sick cell is 1/10⁴When above, the treatment prompt unit prompt treatment is not perfect and needs to carry out other control Treat or improve treatment intensity.

157. systems according to any one of embodiment 154-156, wherein when blood white in subject detected The ratio of sick cell is 1/10⁴Above and 1/10³When following, the treatment prompt unit prompt can carry out bone to shown subject Implantation of marrow operation.

158. systems according to any one of embodiment 154-157, wherein when blood white in subject detected The ratio of sick cell is 1/10³When above, the treatment prompt unit prompt is unsuitable for carrying out bone-marrow transplantation to the subject Operation, and should continue to carry out the subject other treatments or raising treatment intensity.

159. systems according to embodiment 158, wherein other treatments are selected from the drug candidate screening module The monitoring unit prompts for the effective drug candidate, or the therapy similar with its function and/or effect and/or drug.

It is not intended to be limited by any theory, following embodiments is just for the sake of the device of explaination the application, method and is The working method of system, rather than the range of limitation the present application.

Embodiment

Embodiment A modeling module: the specific humanized disease model of leukaemic is established

The leukaemia cell of embodiment A1 stored frozen leukaemic:

It has collected about and the sample of bone marrow of 4 milliliters of leukaemics (wherein leukaemia cell accounts for 90% or more), and is training It supports in base and is prepared for 5 milliliters of cell suspensions, carry out density gradient centrifugation separation with Ficoll.It is outstanding in the cell during this 5 milliliters of Ficoll monocyte separation mediums inject in the lower layer of liquid, and are centrifuged 30 minutes at room temperature, with 800g.After centrifugation, carefully The cell of middle layer is collected, and is transferred into RPMI cell culture medium, leukaemia cell's suspension is obtained.Later, this is thin Born of the same parents' suspension at room temperature, 300g be centrifuged 8 minutes.With RPMI cell culture medium clean precipitating cell, and at room temperature, 300g from The heart 8 minutes, remove supernatant.Obtained cell is suspended in the fetal calf serum containing about 10%DMSO, and is prepared as thin Born of the same parents' density is about 2x10⁷/ ml to about 5x10⁷The cell suspension of/ml.The cell freezing for not preparing to use immediately is stored in liquid nitrogen.

The preparation of embodiment A2 leukaemia cell

The leukaemia cell of stored frozen in embodiment A1 is quickly dissolved at 37 DEG C, and passes through RPMI titration at a slow speed, The cell of freezing is suspended in RPMI culture medium, thus from the leukaemia cell prepared in recovery embodiment A1 in liquid nitrogen. At room temperature, 180g is centrifuged 8 minutes, removes supernatant, the cell being enriched with.The cell one is cleaned with sterile PBS buffer solution It is secondary, and at room temperature, 80g be centrifuged 8 minutes.Then, preparing cell concentration with PBS buffer solution is 4x10⁷The cell suspension of/ml.

Embodiment A3 removes the T cell in cell suspension

Using the magnetic bead of Miltenyi company(i.e. T is thin for the CD3 positive cell for removing in the cell suspension Born of the same parents).By flow cytometry analysis, the CD3 positive T cell in blood sample to Leukemia Patients is detected, such as Fig. 1 institute Show, the upper right corner shows that the CD45/CD3 in sample containing 22.6% is total to positive cell.The institute that will be prepared in 90 microlitres of embodiment A2 It states separated leukaemia cell's suspension and 10 microlitres is coated with CD3 antibodyMagnetic bead (production of Miltenyi company, Sequence number: 130-050-101) it is incubated for 1 hour altogether.Make the screening pipe for co-culturing the culture solution obtained and passing through highfield, from And the cell of the CD3 positive is strapped in screening pipe by the highfield.The cell for passing through the screening pipe is collected, as Eliminate leukaemia cell's sample from patient of CD3 positive T cell.As a result as shown in Figures 2 and 3, wherein Fig. 2 is shown Leukaemia cell eliminating CD3 positive T cell, passing through from the screening pipe.Fig. 3, which is shown, to be trapped in screening pipe CD3 positive T cell.

Processing of the embodiment A4 to immune-deficient mice

It is NSG mouse for the acute lymphoblastic leukemia ALL deficient mice selected, for acute myeloid leukaemia The deficient mice that AML is selected is MISTRG mouse.X-ray irradiation, exposure dose are carried out respectively to two kinds of mouse Are as follows: NSG mouse is 250cGy, and MISTRG mouse is 600cGy.Irradiation uses the X-ray of 4MV, and exposure intensity is 325cGy/ points Clock.12-24 hour before carrying out cell inoculation carries out x-ray irradiation to the mouse, after irradiation, by the mouse It returns in rearging cage.

Embodiment A5 leukaemia cell inoculation and mouse growth condition monitoring

In no-special pathogen (SFB) grade mouse room, immune system defect described in captive breeding embodiment A4 Mouse selects mouse of the age between 5-7 weeks and is inoculated with.Before inoculation, with light irradiation outside 175 tile reds and mouse is heated, and The leukaemia cell for eliminating CD3 positive T cell prepared in embodiment A3 is inoculated into the mouse by tail vein injection (inoculum concentration is shown in Table 1) in vivo monitors the behavior situation of mouse daily after inoculation.Since after inoculation 2-3 weeks, flow cytometer is used HuCD45 is monitored weekly⁺CD19⁺Ratio of the cell in the mouse peripheral blood through being inoculated with, to monitor leukaemia cell in mouse Intracorporal implantation and growth conditions.For the cell in different patient sources, from date difference is monitored, as shown in table 1.By Table 1 successfully constructs leukaemia humanization disease model as it can be seen that in each sample.

Table 1

Leukaemia cell of the embodiment A6 from mouse spleen separation humanization

HuCD45 in the peripheral blood of the disease model described in the embodiment A5⁺When cell reaches 50% or more (referring to Upper table), by the model mice sacrificed by carbon dioxide.The spleen that the mouse is separated with sterile surgical scissors, is placed in RPMI In culture medium.Mouse spleen is placed in sterile metal screen and is pulverized, and collects fragment of tissue in RPMI culture medium.It uses The plastic filter screen that aperture is 40 microns, further pulverizes spleen tissue fragment, 30 milliliters of cell suspensions is prepared.In the cell 15 milliliters of lymph separating liquids (Ficoll solution) are injected by the lower layer of suspension, and at room temperature, 800g is centrifuged 30 minutes.Careful collection Intermediate layer cell (cellular layer for being enriched with leukaemia cell), and be transferred into RPMI cell culture medium.It will be obtained Leukaemia cell's suspension at room temperature, 300g be centrifuged 8 minutes.The cell through precipitating is cleaned with RPMI cell culture medium, and in room Under temperature, 300g is centrifuged.The cell precipitation obtained after centrifugation is suspended in the fetal calf serum containing 10%DMSO, and is prepared It is 2x10 for cell density⁷-5x10⁷The cell stock solution of/mL, its stored frozen is spare in liquid nitrogen.

Application of the specific humanized disease model of embodiment B leukaemic in drug candidate screening

Embodiment B1 assesses pharmaceutical efficacy using leukaemia humanization disease model

As huCD45 in the peripheral blood of constructed model mice⁺When cell reaches 1-5%, which is grouped at random In vehicle control group and administration group, every group of 6 mouse, application drug candidate is treated.Selected exemplary candidate drug And administration time corresponding to drug, dosage and administration mode are as follows:

Vincristine: intraperitoneal injection, 0.5mg/kg, once a week, it is for 4 weeks；

Ninopterin: intraperitoneal injection, 5mg/kg, once a day, every other week injection continue 8 weeks；

Dexamethasone: intraperitoneal injection, 15mg/kg, once a day, it is for 4 weeks；

Adriamycin: intravenous injection, 1.5mg/kg, once a week, it is for 4 weeks；

Left-handed asparagine: intraperitoneal injection, 1000KU/kg, once a day, it is for 4 weeks.

After applying drug candidate to model mice, huCD45 is monitored weekly with flow cytometer⁺And/or CD19⁺Cell is in institute The variation of proportion in mouse peripheral blood cell is stated, and the mouse of the vehicle control group and administration group is compared, from And drug candidate is understood to the effect of leukemia treating.

In the present embodiment, the evaluation criteria for pharmaceutical efficacy is as shown in figure 4, leftmost curve part is the white blood of people in Fig. 4 Ratio of the sick cell during administration in mouse peripheral blood, right block are shown according to drug candidate to people's cell (huCD45⁺ And/or CD19⁺) ratio influence, drug is divided into six grades.As it can be seen that only when drug kills leukaemia in Mice Body Cell, and when making its ratio keep below 1%, and maintain 2 weeks or more, it is believed that the drug is makes disease complete incidence graph.

Verifying of the embodiment B2 to the application disease model clinical correlation

In the present embodiment, by taking dexamethasone as an example, the clinical correlation of the disease model of the application is demonstrated, as a result such as table Shown in 2.The state of an illness is cured or alleviates, i.e. it is to face that leukaemia cell of the patient after the treatment in blood disappears completely or partially The important indicator of pharmaceutical efficacy is evaluated on bed.As shown in table 2, quick relative to drug candidate for the patient of clinically drug resistance group Sense group, complete incidence graph duration are short.Correspondingly, in corresponding disease model, delay completely after applying drug candidate The duration of solution is also shorter.

Table 2

Then, genome analysis also has been carried out to the cell in the disease model of the application.Briefly, to medicaments insensitive group The analysis of the gene expression of full-length genome has been carried out with the leukaemia cell being originated from the disease model of patient described in drug resistance group, As a result as shown in Figure 5.It (is clinically shown to from (clinically the showing drug resistance) patient of leukaemia -50 and leukaemia -54 first Drug susceptibility) disease model of the application of patient carried out internal dexamethasone drug study (Fig. 5 A and B), proved recipe in fact Method and formulation rate are as described in Example 7.In addition, to the leukaemia cell for being originated from this two patients of leukaemia -50 and leukaemia -54 It has carried out in vivo cytotoxicity experiment (Fig. 5 C), method is alma indigo plant fluorescence developing method.These results show to be detected Drug be consistent in the effect in disease model described herein with the effect in the patient that the model is originated from.

Hereafter, the cell in disease model described herein is carried out with the cDNA microarray that full genome is expressed Gene expression analysis.Briefly, dexamethasone is applied after application 8 hours to the disease model and extracts the intracorporal white blood of mouse Sick cell separates RNA, obtains cDNA by reverse transcription, and cDNA is placed on cDNA microarray and carries out complete or collected works because of expression Analysis.The result is compared with the control group result not being administered, has obtained the information of the gene expression of induced by dexamethasone. In figure 5d, the reliability of data is confirmed by principal component analysis (PCA) first；Then compared medicaments insensitive and drug resistance Disease model whether there is or not dexamethasone (Dex) administration under conditions of expression conditions (show only part representativeness base Cause, Fig. 5 E), it is seen then that same gene in both models expression and administration before and after express be changed significantly not Together.In turn, by the lab topics to the application's for being originated from matched 5 sensitive group patients of genotype and 5 drug resistance group patients Disease model (Fig. 5 F), it is seen then that the variation of expression conditions is similar between the disease model of medicaments insensitive, drug resistant disease model Between gene expression the case where it is also similar, and the variation of expression conditions is entirely different between these two types of model.

In addition, the cDNA microarray also expressed with full genome is in the cell of patient and the disease model by its building Cell compared (Fig. 5 G), and according to full genome express data carry out hierarchical cluster (Hierarchy Clustering bioinformatics research), it was demonstrated that the genome of the cell from patient and constructed corresponding disease mould The genome of leukaemia cell in type exactly matches.In Fig. 5 G, P represents patient's primary cell in patient code, and X1 represents one The cell obtained after secondary modeling, X2 represent the cell obtained after two modelings.

Embodiment C detects the presence and/or ratio of leukaemia cell in the subject

The amplification and identification of embodiment C1 acute lymphoblastic leukemia (ALL) subject's target gene

The acquisition of C1.1 subject DNA

It is diagnosed with before subject's reception treatment of acute lymphoblastic leukemia, obtains tested from this at every The sample (for example, blood cell samples, or the DNA sample extracted from the cell) of person, and the sample is serially diluted (for example, 10 times, 100 times, 1000 times, 10,000 times, 100,000 times or more of dilution).After the subject is subjected to the treatment, The sample (for example, blood cell samples, or the DNA sample extracted from the cell) for being originated from the subject is obtained again.

For every subject, collect every time about 2 milliliters of its sample of bone marrow (in the sample of bone marrow, leukaemia cell's ratio At least 90%) example is.It is prepared for the cell suspension of 5 milliliters of samples in culture medium, and carries out density gradient centrifugation with Ficoll Separation.During this, the cell suspension lower layer inject 5 milliliters of Ficoll lymphocyte separation mediums, and at room temperature, with 800g is centrifuged 30 minutes.After centrifugation, the cell of careful collection middle layer, and be transferred into RPMI cell culture medium, it obtains Leukaemia cell's suspension from subject.

Later, by leukaemia cell's suspension at room temperature, 300g be centrifuged 8 minutes.It is heavy to be cleaned with RPMI cell culture medium The cell in shallow lake, and at room temperature, 300g be centrifuged 8 minutes, remove supernatant.Obtained cell is suspended in containing about 10%DMSO's In fetal calf serum, and being prepared as cell density is about 2x10⁷/ ml to about 5x10⁷The cell suspension of/ml.It will not prepare immediately The cell freezing used is stored in liquid nitrogen.

With 20 kit of NucleoBond CB (it is purchased from NucleoBond company, is carried out according to the specification in kit Operation) DNA in cell suspension is separated to get the DNA of the subject.As needed, to subject's DNA sample obtained It is diluted.

The amplification of target gene in C1.2 subject

(1) target gene includes: the coding region of IgH, IgK, IgL, TCRA/D, TCRB and TCRG.

(2) PCR reaction system used in are as follows: 10 μ L amplification buffers, 4 kinds of dNTP each 200 μm of ol/L, 100pmol/L Primer, 2 μ g templates (DNA from subject obtained in embodiment C1.1), 2.5 μ g Taq DNA polymerases, 1.5mmol/ L Mg²⁺With the distilled water for complementing to 100 μ L volumes.Wherein, reagent needed for carrying out PCR reaction is purchased from Takara company.

(3) PCR reaction condition is as shown in the table:

(4) it is directed to above-mentioned different target gene, different primer combinations is respectively adopted and carries out PCR:

A), for the sample from B cell acute lymphoblastic leukemia (B-ALL) subject, 24 groups of primers are used altogether Combination, in which:

To target gene IgH, combined using primer as shown in table 3:

Table 3

	Forward primer title	Reverse primer title
			1st group	VH1/7	JH cons
2nd group	VH2	JH cons
			3rd group	VH3	JH cons
4th group	VH4	JH cons
			5th group	VH5	JH cons
6th group	VH6	JH cons
			7th group	DH1/4/5/7	JH cons
8th group	DH2	JH cons
			9th group	DH3	JH cons
10th group	DH6	JH cons

To target gene IgK, combined using primer as shown in table 4:

Table 4

To target gene TCR, combined using primer as shown in table 5:

Table 5

B), for the sample from T cell acute lymphoblastic leukemia (T-ALL) subject, 16 groups of primers are used altogether Combination, in which:

To target gene TCR, combined using primer as shown in table 6:

Table 6

C), the specific nucleotide sequence of above-mentioned primer is as shown in table 7:

Table 7

The separation of the pcr amplification product of C1.3 target gene:

Gel electrophoresis separation is carried out to the resulting pcr amplification product of embodiment C1.2, wherein using 2% (w/w) agarose Gel electrophoresis 2 hours at voltage 100V.

Fig. 7 reflects the result that the gel electrophoresis of amplification target gene IgH is combined using VH3 and JH con primer.Fig. 7 In, positive controls are the cell strain using known IgH rearrangement (for example, B cell non-Hodgkin lymphoma (B-NHL) cell Strain), the DNA extracted according to embodiment C1.1 carries out the amplified production of PCR reaction as template, occurs one in 400bp or so A obvious and clearly DNA band, and in 4 subjects of display, the 2nd occur with the 3rd subject it is similar DNA band.This illustrates that this band is with specificity for this two subjects.

Similarly, the gel electricity of the pcr amplification product of amplification acquisition is carried out for 30 target gene regions by comparing Swimming analysis result (including 10 regions IgH shown in the application table 1-4,4 regions IgK and 16 regions TCR), obtains For every subject, (i.e. the characteristic of the leukaemia cell of the subject expands produces specific pcr amplification product Object).

Since leukaemia is generally monoclonicity disease, all leukaemia cells are substantially all derived from a gene The cell clone of variation.Diversity based on cell, normal person is not in the immunoglobulin rearrangement of specificity, and leukaemia Cell mass but shows as the specificity to specific gene rearrangement, i.e., the specific region occur massive amplification, show it is specific by The DNA cloning product band of examination person's specificity.

Embodiment C2 analyzes the characteristic amplified production of the leukaemia cell of subject

C2.1 gene sequencing

The characteristic amplified production of each subject leukaemia cell identified in recycling embodiment C1.3, then uses The method of " GeneScanning " carries out gene sequencing to the characteristic amplified production of the subject leukaemia cell of acquisition, obtains The specific nucleotide sequence of the blood disease cells characteristic amplified production of each subject.

The concrete operation step of " GeneScanning " is referring to document Linke B, Bolz I, Fayyazi A, von Hofen M,Pott C,Bertram J et al.Automated high resolution PCR fragment analysis for identification of clonally rearranged immunoglobulin heavy chain genes.Leukemia 1997；The record of the part " Cloning and DNA sequencing " in 11:1055-1062..

The interpretation of result of C2.2 gene sequencing

It is each tested by what is obtained in embodiment C2.1 using BLAST tool (referring to blast.ncbi.nlm.nih.gov) There is no the normal person of cases with leukemia to be had in the nucleotide sequence and ncbi database of person leukaemia cell's characteristic amplified production The nucleotide sequence of some correspondence target genes is compared, and produces to identify each subject leukaemia cell's characteristic amplification In the nucleotide sequence of object, which is conservative sequence (existing in other subjects), which is subject's leukaemia The characteristic gene sequence of cell (that is, can be used for characterizing and identifying the leukaemia cell of the subject, and is substantially not present in it In its subject or in non-leukaemia cell).

By taking the fragment length recycled in Fig. 7 from the 2nd subject is the characteristic amplified production of 400bp as an example, BLAST Result it is as shown in Figure 8.In Fig. 8, the sequence (existing in other subjects) that the sequence expression in frame is guarded, and two Nucleotide sequence between frame is the characteristic gene sequence of the leukaemia cell of subject.As can be seen from FIG. 8, subject The characteristic gene sequence of leukaemia cell is ACGGGGGCCGTAG.

Analysis of the embodiment C3 to the characteristic gene sequence of leukaemia cell

C3.1 detects the qPCR of subject leukaemia cell's characteristic gene sequence

(1) based on each subject leukaemia cell for analyzing acquisition in embodiment C2.2 characteristic gene sequence (for example, As exemplary gene order ACGGGGGCCGTAG), the individuation amplimer that design specific can expand it Group.

(2) fluorescent quantitative PCR reaction is carried out

By primer designed in step (1), with the probe and specificity for specifically binding non-characteristic gene sequence The primer sets for expanding non-characteristic gene sequence are combined, i.e., using the characteristic base for being directed to particular subject leukaemia cell (such as because of the primer of sequence design, suitable for the non-characteristic gene amplimer (as shown in table 8) of all subjects and fluorescence probe Shown in table 8) carry out qPCR reaction.In qPCR, having a primer in each pair of primer is be specific to specific subject white The characteristic gene sequence of blood disease cell, and another primer is the non-characteristic gene for all subjects, and Used fluorescence probe is also the non-characteristic gene for all subjects.

Table 8

(3) qPCR reaction system are as follows: 2 μ L DNA profiling (the subject leukaemia cells obtained in embodiment 1.1 DNA), 0.3 μ L forward primer, 0.3 μ L reverse primer, 0.4 μ L fluorescence probe, 10 μ L Taqman Universal PCR Master Mix and 7 μ L dH₂O。

(4) qPCR reaction condition is as shown in the table:

(5) qPCR reaction result

(for example, 10 times, 100 times, 1000 times, 10,000 times or 100,000 times of dilution) is serially diluted with what is obtained in step (1) Treatment before subject's DNA sample as template, according to as described in this embodiment, qPCR reaction is carried out respectively, to build Found a series of standard amplification curves corresponding to variant DNA sample concentration.

In addition, the DNA sample of the subject is as template after the treatment also obtained using in step (1), according to the present embodiment Described in carry out qPCR reaction, measurement result after being treated.By by measurement result after the treatment and the standard Amplification curve is compared, to judge the ratio of leukaemia cell's characteristic gene sequence in the subject after treatment, And judged after treatment accordingly, the ratio A of leukaemia cell in the subject.

With with IgH primer in table 6 (GCGCGRAACAGGTACTGG (SEQ ID NO:126) and its correspondent probe TCTCCTATACTACACGCTCT) the patient-specific primer of (SEQ ID NO:175) and the 2nd subject (CCGTGGTGTCACA (SEQ ID NO:222)) to Exemplary gene sequence ACGGGGGCCGTAG described in embodiment C2 into For the result of row qPCR amplification, as a result as shown in Figure 9.The DNA sample of the subject is as template after receiving treatment 15 days, The arrow of " the 15th day " is signified in qPCR result obtained such as Fig. 9, and the result and series standard curve (are made respectively in Fig. 9 It is 1/10,1/10 with leukaemia cell's DNA ratio²、1/10³、1/10⁴、5/10⁵Sample as template, carry out the present embodiment and retouch The qPCR reaction stated is obtained as a result, being respectively labeled as 10^-1、10^-2、10^-3、10^-4And 5x 10^-5) be compared, it can be with See, after treatment in 15 days, the ratio for the leukaemia cell for including in subject's body is about 1/10²。

Similarly, using method and system described in this application to the residual leukemic cell ratio in all subjects Example is detected.

The interpretation of result of C3.2qPCR detection

The ratio A of leukaemia cell can be used for instructing subsequent therapeutic scheme in the subject measured in embodiment C3.1 (as shown in table 9): in A > 1/10⁴In the case where, can further analyze the size of the ratio A, thus judge whether be suitable for pair The subject carries out bone-marrow transplantation.For example, if 1/10⁴<A<1/10³, can be shown that can carry out bone-marrow transplantation to the subject Operation；If A > 1/10³, can be shown that there are a large amount of leukaemia cells in subject's body, due to higher leukaemia cell Residual degree can make a possibility that leukemia relapse greatly increase, thus be considered as in this case the subject be unsuitable for into Row bone marrow transplant, and should continue to receive the treatment of a new round or improve the intensity for the treatment of

Table 9

As it can be seen that according to Fig. 9's as a result, the subject should also continue to receive treatment or improve the intensity for the treatment of.

Figure 10 and Figure 11 can further illustrate the leukaemia diagnosis and treatment described herein that the above embodiments A-C is reflected System.Specifically, Figure 10 is the schematic diagram of one example of operating process of herein described leukaemia diagnosis and therapy system.For example, by Examination person is diagnosed as after removing hospital admission with leukaemia, passes through the modeling module of the leukaemia diagnosis and therapy system, building at this time The specific humanized disease model of subject；Hereafter subject receives this field routine, standardized treatment, when the treatment knot Shu Hou, then by the detection module of the leukaemia diagnosis and therapy system, depositing for subject leukaemia cell is detected on a molecular scale And/or ratio；If the result of detection illustrates that the subject has biggish probability that can recur leukaemia or leukaemia cell Ratio it is excessively high, then illustrate subject to it is above-mentioned it is conventional, it is standardized treatment produce drug resistance；It can be seen that such In the case of, need specificity in the active drug of subject, therefore screen using the drug candidate of the leukaemia diagnosis and therapy system Module, using the specific humanized disease model screening drug candidate of subject (chemotherapeutic, targeting medicine), according to previously described The level results of drug candidate choose the drug candidate guiding clinical treatment of class 5 or class 6, receive the subject special Property in itself screening drug treatment；After treating a period of time, if by the detection module point of the leukaemia diagnosis and therapy system Show that the ratio of detection subject leukaemia cell is very low or does not have at all in sub- level, then it is assumed that the leukaemia diagnosis and therapy system Screening drug obtained is very significant to the curative effect of the subject.

Figure 11 is then the schematic diagram of an example of the relationship of each functional unit in herein described leukaemia diagnosis and therapy system. The leukaemia diagnosis and therapy system can contain modeling module, detection module and the big module of drug candidate screening module three, wherein detecting Module can contain 2 or more.Each module plays the unit of different function containing several.

Specifically, the modeling module may include the sample comprising leukaemia cell's suspension from the subject Unit, the radiating element comprising radioactivity processing unit and for by leukaemia cell's inoculation of suspension liquid to through the radiation The inoculation unit of the immune-deficient mice of cell processing.

The modeling module can also include the separative unit that can separate Samples subjects.At through the sample unit Before reason, it can be handled through the separative unit.The modeling module can also include that removal is originated from non-T cell type leukaemia subject Sample in T cell to obtaining the T cell removal unit of leukaemia cell's suspension.At through the sample unit Before reason, alternatively, can be removed through the T cell single after separative unit processing and before being handled through the sample unit Member processing.The modeling module can also include the infrared radiation and heating unit being irradiated using infrared light supply to individual. Before being handled through the radiating element, it can be handled through the infrared radiation and heating unit.The modeling module can also include The leukaemia cell from the subject is monitored in the prison of the intracorporal growing state of the immune-deficient mice through being inoculated with Survey unit.After the inoculation cell processing, can be handled through the monitoring unit.The modeling module can also include to institute State the spleen processing unit that the spleen of disease model is handled.After the inoculation cell processing, alternatively, connecing described in the warp After kind cell processing and before being handled through the monitoring unit, it can be handled through the monitoring unit.The modeling module can be with Including liquid nitrogen.After inoculation monitoring unit processing, can be handled through liquid nitrogen.

The detection module may include specific amplification target gene to obtain the amplification of target specificity amplified production Unit.The detection module, which may also include to identify, is originated from the white of the subject in the target specificity amplified production The identification unit of the characteristic amplified production of blood disease cell.Through the amplification unit processing after, can be through the identification unit at Reason.The detection module may also include the characteristic gene sequence that can obtain the leukaemia cell for being specific to the subject Analytical unit.Through the amplification unit processing after, can be through the identification cell processing.The detection module may also include can Judge the recognition unit of the presence of residual leukemic cell and/or ratio in the subject.It is handled through the analytical unit It afterwards, can be through the recognition unit processes.The detection module may also include can be thin according to leukaemia in subject detected The treatment prompt unit for the successive treatment scheme that the presence of born of the same parents and/or ratio prompt use corresponding subject.Through the knowledge After other cell processing, it can be handled through the treatment prompt unit.

The drug candidate screening module may include the first drug candidate group containing one or more drugs, containing one kind Or the second drug candidate group of a variety of drugs.The drug candidate screening module can also include monitoring from the subject's Monitoring unit of the leukaemia cell in the intracorporal growing state of the immune-deficient mice through being inoculated with.Through applying described the After one drug candidate group and the second drug candidate group, it can be handled through the monitoring unit.

Aforementioned detailed description is to be not intended to limit scope of the appended claims to explain and provide with example way. A variety of variations of current embodiment cited herein are it will be apparent that and retaining for those of ordinary skills In the range of the attached claims and its equivalent program.

Sequence table

<110>Hangzhou He Ma Biotechnology Co., Ltd

<120> 0022-PA-004

<130>system for carrying out leukaemia diagnosis and treatment

<160> 222

<170> PatentIn version 3.5

<210> 1

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> VH1/7

<400> 1

tctccccctc tcctctacta 20

<210> 2

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH1/7

<400> 2

cccctcagtc tacctctcct gcaag 25

<210> 3

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH1/7

<400> 3

ctccgtgcct cacccccctg ctcaa 25

<210> 4

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> VH2

<400> 4

accttctagc tgtctcctcc t 21

<210> 5

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH2

<400> 5

gtctcctcct acgctcctct aaccc 25

<210> 6

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH2

<400> 6

tgcttccgtc agcccccacc ctacc 25

<210> 7

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> VH3

<400> 7

ccccgtccct ctctctctc 19

<210> 8

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH3

<400> 8

ctcccccctc cctctctctc tcctg 25

<210> 9

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> VH3

<400> 9

cctccgccac cctccaccct a 21

<210> 10

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223> VH4

<400> 10

gcccagctct cctctagc 18

<210> 11

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH4

<400> 11

cttcgctctc cctgtccctc acctg 25

<210> 12

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH4

<400> 12

tgcttccgcc agcccccacc ctacc 25

<210> 13

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> VH5

<400> 13

ctcctgcagt ctgctgcag 19

<210> 14

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH5

<400> 14

cccgctgtct ctctacttct cctgt 25

<210> 15

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH5

<400> 15

ccgtgcgcca cttgcccccc taacc 25

<210> 16

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> VH6

<400> 16

gtacagctgc agcagtcacc t 21

<210> 17

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH6

<400> 17

tcgcactccc tctcactcac ctgtg 25

<210> 18

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> VH6

<400> 18

tgcttcaccc agtccccatc ctctg 25

<210> 19

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> JH cons

<400> 19

acctctgctc tccctctcc 19

<210> 20

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> JH cons

<400> 20

cttacctctg ctctccctct cc 22

<210> 21

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> DH1

<400> 21

ayccagctcc ccccactgcw ca 22

<210> 22

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223> DH1

<400> 22

cccgctatgt gtgcaccc 18

<210> 23

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> DH2

<400> 23

cagcactccg ctcactgtcc tctc 24

<210> 24

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> DH2

<400> 24

gcactccgct cactgtcctc t 21

<210> 25

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> DH3

<400> 25

cctcctcmcc tcagcccygc tcat 24

<210> 26

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> DH3

<400> 26

gtccccctcc ctatataaaa 20

<210> 27

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> DH4

<400> 27

cccagctcgc accrctgtca a 21

<210> 28

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> DH4

<400> 28

acttccccag ctcgcagca 19

<210> 29

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> DH5

<400> 29

acccagcctg ctctccactg 20

<210> 30

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> DH5

<400> 30

caccccctca ctgtgcatgt 20

<210> 31

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> DH6

<400> 31

cacccccccc araaccagkg wt 22

<210> 32

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> DH6

<400> 32

tctccccagc aaccctacc 19

<210> 33

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> DH7

<400> 33

ccgctcccct ctcccacgtg tttt 24

<210> 34

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223> DH7

<400> 34

cacacccccc ctaccagc 18

<210> 35

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vk1

<400> 35

gtagctctca ctgtcaccat cact 24

<210> 36

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Vk1

<400> 36

tcaaccttca gccccagtgc ttctg 25

<210> 37

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vk2

<400> 37

tgctctgccc ccctccatct c 21

<210> 38

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223> Vk2

<400> 38

cccctccatc tcctgcacct ctagtc 26

<210> 39

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vk3

<400> 39

ccctaactgc caccctctcc tg 22

<210> 40

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Vk3

<400> 40

cccaccctcc tcatctatct tgcatcc 27

<210> 41

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vk4

<400> 41

cccctctccg ccaccatcaa c 21

<210> 42

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Vk4

<400> 42

caactgcaag tccagccact gtgtttt 27

<210> 43

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Intron RSS

<400> 43

gttattccca aaagctcaat ctcaaag 27

<210> 44

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Intron RSS

<400> 44

cgtcccaccg cctgctgtac tc 22

<210> 45

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Kdel

<400> 45

cccttcatac tcccttcacc cac 23

<210> 46

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Kdel

<400> 46

cctcactcct cactgcacct tgtccta 27

<210> 47

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vg1

<400> 47

caccccctct ccgtcatctg c 21

<210> 48

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> Vg1

<400> 48

gctacccccc acagcrtctt 20

<210> 49

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vg2

<400> 49

cagcccgcct gctatgtgtc c 21

<210> 50

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> Vg2

<400> 50

agcatccgta actcaagcaa 20

<210> 51

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vg3

<400> 51

ctcatacctt gcaacttatc ctgc 24

<210> 52

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vg3

<400> 52

ccccactgtc actaagctat c 21

<210> 53

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Vg4

<400> 53

ctctaatatc tatttccact ccagc 25

<210> 54

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> Vg4

<400> 54

cttccacttc cactttctaa 20

<210> 55

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Jg1 (Jg1.1/2.1)

<400> 55

ttaccacctc tagttactat ctgc 24

<210> 56

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Jg1 (Jg1.1/2.1)

<400> 56

ttaccacccc tagttactat ctgc 24

<210> 57

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Jg2 (Jg1.3/2.3)

<400> 57

ccgtatatgc acaaagccaa atc 23

<210> 58

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Jg2 (Jg1.3/2.3)

<400> 58

gtgttgttcc actgccaaac tg 22

<210> 59

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vd1

<400> 59

actaccgccc agtcatcagt atcc 24

<210> 60

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vd1

<400> 60

atgcaaaaag tcctcgctat t 21

<210> 61

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Vd2

<400> 61

accaaacagt gcctgtgtca atacc 25

<210> 62

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Vd2

<400> 62

ataccctcta aagctcatct atg 23

<210> 63

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vd3

<400> 63

ctccactccc tcccctgtcc c 21

<210> 64

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vd3

<400> 64

gtaccgctta accccacttt a 21

<210> 65

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Dd2

<400> 65

actccatgtt caaatactta tagtatt 27

<210> 66

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Dd2

<400> 66

agcccgtcct cttcccaaag t 21

<210> 67

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Dd3

<400> 67

ctaatcccac ttttgcccct gcag 24

<210> 68

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Dd3

<400> 68

tccctcccac cgtctgctta t 21

<210> 69

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jd1

<400> 69

acctcttccc agctgtcctc c 21

<210> 70

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jd1

<400> 70

gttccacagt cacacccgtt c 21

<210> 71

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Ja9

<400> 71

tttaactccc actcaaaact atg 23

<210> 72

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Ja29

<400> 72

cccaaaagca ttctacctac a 21

<210> 73

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223> Ja30

<400> 73

gccacccaca tgtcttag 18

<210> 74

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> Ja48

<400> 74

tccccactat cttatgcag 19

<210> 75

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> Ja49

<400> 75

gcagtttaaa ccgtttgct 19

<210> 76

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223> Ja52

<400> 76

ccgctaccct gcaaaag 17

<210> 77

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Ja54

<400> 77

ctcccccaag taattaaatc a 21

<210> 78

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Ja55

<400> 78

agtatacgtc cctcaagctc t 21

<210> 79

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Ja56

<400> 79

agctctttcc cttatctttc a 21

<210> 80

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Ja57

<400> 80

gctcctccgt tataaaacac t 21

<210> 81

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223> Ja58

<400> 81

ctcttctatg tcccactct 19

<210> 82

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> Ja59

<400> 82

atcaaatcct caccctctag 20

<210> 83

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Ja61

<400> 83

gtttgttaac ccacattact atc 23

<210> 84

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Sildb

<400> 84

aaccgctgct agtccctcta a 21

<210> 85

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223> Taldb1

<400> 85

actgcctgtc gccaacta 18

<210> 86

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb2

<400> 86

aactatgttt tcctatcgtc a 21

<210> 87

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vb4

<400> 87

caccttgttc tcctaccgtc agca 24

<210> 88

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb5/1

<400> 88

cagtgtgtcc tcctaccaac ag 22

<210> 89

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb6a/11

<400> 89

aaccctttat tcctaccctc a 21

<210> 90

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb6b/25

<400> 90

atcccttttt tcctaccaac ag 22

<210> 91

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb6c

<400> 91

aaccctttat tcctatcaac ag 22

<210> 92

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb7

<400> 92

cgctatgtat tcctacaagc a 21

<210> 93

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Vb8a

<400> 93

ctcccgtttt ctcctacact cactc 25

<210> 94

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb9

<400> 94

cgctatgtat tcctataaac ag 22

<210> 95

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223> Vb10

<400> 95

ttatgtttac tcctatcgta actagc 26

<210> 96

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb11

<400> 96

caaaatgtac tcctatcaac aa 22

<210> 97

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Vb12a/3/13a/15

<400> 97

atacatgtac tcctatcctc aactc 25

<210> 98

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223> Vb13b

<400> 98

ccccatgtac tcctatactc aag 23

<210> 99

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vb13c/12b/14

<400> 99

gtatatgtcc tcctatcctc aact 24

<210> 100

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223> Vb16

<400> 100

taacctttat tcctatcctc gtgt 24

<210> 101

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb17

<400> 101

ccccatgtac tcctaccctc a 21

<210> 102

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb18

<400> 102

tcatgtttac tcctatcccc ag 22

<210> 103

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223> Vb19

<400> 103

ttatgtttat tcctatcaac actatca 27

<210> 104

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb20

<400> 104

caacctatac tcctaccctc a 21

<210> 105

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Vb21

<400> 105

taccctttac tcctaccccc ag 22

<210> 106

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223> Vb22

<400> 106

atacttctat tcctacactc aaatct 26

<210> 107

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb23/8b

<400> 107

caccctctac tcctaccagc a 21

<210> 108

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Vb24

<400> 108

cgtcatgtac tcctaccagc a 21

<210> 109

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Db1

<400> 109

gccaaacagc cttacaaact c 21

<210> 110

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223> Db2

<400> 110

tttccaagcc ccacacagtc 20

<210> 111

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.1

<400> 111

cttacctaca actgtctatc tcctg 25

<210> 112

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.2

<400> 112

cttacctaca acccttaacc tcctc 25

<210> 113

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.3

<400> 113

cttacctaca acagtctgcc aactt 25

<210> 114

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.4

<400> 114

catacccaac tcactctgct ccgttc 26

<210> 115

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.5

<400> 115

cttacctagc ttgctctgtc ctgtc 25

<210> 116

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Jb1.6

<400> 116

catacctgtc acagtctgcc tg 22

<210> 117

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.1

<400> 117

ccttcttacc tagcaccctc t 21

<210> 118

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.2

<400> 118

cttacccagt accctcagcc t 21

<210> 119

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.3

<400> 119

cccgcttacc ctgcactgtc a 21

<210> 120

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.4

<400> 120

ccagcttacc cagcactctc t 21

<210> 121

<211> 16

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.5

<400> 121

cgcgcacacc ctgcac 16

<210> 122

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.6

<400> 122

ctcgcccagc accctcagcc t 21

<210> 123

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223> Jb2.7

<400> 123

cttacctgta accgtctgcc tg 22

<210> 124

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 124

ctagaaycag gcctgrgtca c 21

<210> 125

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 125

ctactctcta ctcctgcgtc 20

<210> 126

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 126

gcgcgraaca ggtactgg 18

<210> 127

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 127

ggctcaactg tcctgtyagg 20

<210> 128

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 128

gtccttaaag gtacaaggca g 21

<210> 129

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 129

ccgttagggc tctcacctct 20

<210> 130

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 130

agcctctagc taacgctaca aac 23

<210> 131

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>IgH primer

<400> 131

cctctaaaca aagcgggtac tgt 23

<210> 132

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 132

gcctggcgct cgtgtact 18

<210> 133

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 133

gctcacttat cactctcagg taca 24

<210> 134

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 134

cagtgggtca gcctctgtaa a 21

<210> 135

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 135

aggctgcgaa ggtaggctgg 20

<210> 136

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 136

gtacacaact aactcacagg ctacag 26

<210> 137

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 137

atagctcaca aagctacaca aagta 25

<210> 138

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 138

tcacaaaaca acctcaatag taaca 25

<210> 139

<211> 28

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 139

atatctggtc tcgtatatac tcaaacac 28

<210> 140

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 140

ggtacactgg caataaacac tactc 25

<210> 141

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>IgK primer

<400> 141

tacactcagg tggtcactgg tcag 24

<210> 142

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 142

acaaacccta cctcaagcta caac 24

<210> 143

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 143

gctgctaagt tattaactct aacaggc 27

<210> 144

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 144

acaaacgcta cctcaagcta ctact 25

<210> 145

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 145

atacaactta cagtactgct aagttat 27

<210> 146

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 146

cctcaggcta ctcgcaaaat aaa 23

<210> 147

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 147

tctcaggtat cacatagtac tctaaca 27

<210> 148

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 148

ctctcacaac ataggaaagg tacaag 26

<210> 149

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 149

ggtggctact ctaactaaca actgg 25

<210> 150

<211> 28

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 150

aactatactg cttaaaaaac actaacta 28

<210> 151

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>TCRg primer

<400> 151

aaggtcgcaa ggctcttata ggaa 24

<210> 152

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a primer

<400> 152

ccgcaaaagg taaacacgca aac 23

<210> 153

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a primer

<400> 153

aagctaaggc taaaagctac tactg 25

<210> 154

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a primer

<400> 154

ggctggaatc aaactcataa agg 23

<210> 155

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a primer

<400> 155

caaagtagct ggtacgcaaa aggaa 25

<210> 156

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a primer

<400> 156

aacgggctcc tgaaaatgta c 21

<210> 157

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>

TCRb primer

<400> 157

ctctggtggt ctctggcagg 20

<210> 158

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 158

ctacctggct gctaactaaa aa 22

<210> 159

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 159

cgaaaggctc ctgggtcag 19

<210> 160

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 160

cgaaaggctc ctgggtcag 19

<210> 161

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 161

cgaaaggctc ctgggtcag 19

<210> 162

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 162

cgaaaggctc ctgggtcag 19

<210> 163

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 163

cgaaaggctc ctgggtcag 19

<210> 164

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 164

cgaaaggctc ctgggtcag 19

<210> 165

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 165

cgaaaggctc ctgggtcag 19

<210> 166

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 166

caggctcctg ctgctcg 17

<210> 167

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 167

caggctcctg ctgctcg 17

<210> 168

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 168

caggctcctg ctgctcg 17

<210> 169

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 169

caggctcctg ctgctcg 17

<210> 170

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>TCRb primer

<400> 170

caggctcctg ctgctcg 17

<210> 171

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>1 primer of Tal

<400> 171

aaggaactta cgtccgtcaa gt 22

<210> 172

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>1 primer of Tal

<400> 172

ggaacttacg tccgtct 17

<210> 173

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 173

acgctactag ctcgtct 17

<210> 174

<211> 16

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 174

tactgctact agctgg 16

<210> 175

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 175

tctcctatac tacacgctct 20

<210> 176

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 176

tctggctcta cgtctcgt 18

<210> 177

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 177

tctggctcct cgtctact 18

<210> 178

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 178

ggctggtcag ggtctggtca ggtg 24

<210> 179

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 179

caaggctcat aggtcagggt ctcaaca 27

<210> 180

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>IgH fluorescence probe

<400> 180

cacggtcagg gtctggtcag gtaacta 27

<210> 181

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 181

ctcgccgggt caatctcgta tctgt 25

<210> 182

<211> 28

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 182

tggtgtactt acgtcgtagc tgaatcct 28

<210> 183

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 183

acactctaat acacctgggt gctgcgt 27

<210> 184

<211> 33

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 184

acgtcgtaaa aacgctatag ctcttaaagc tgt 33

<210> 185

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 185

aggaatagaa tcctctc 17

<210> 186

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 186

aggtctagaa tcgtatc 17

<210> 187

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 187

caggttaaga atcgtatc 18

<210> 188

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 188

aggtctataa acgtatc 17

<210> 189

<211> 17

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 189

aggtctataa acgtatc 17

<210> 190

<211> 33

<212> DNA

<213>artificial sequence

<220>

<223>IgK fluorescence probe

<400> 190

acgtcgtaaa aacgctatag ctcttaaagc tgt 33

<210> 191

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 191

aactgtatgt aactcgctat a 21

<210> 192

<211> 18

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 192

aggtgctcgt ggtatatg 18

<210> 193

<211> 25

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 193

cctatacaat atctaatact ctctg 25

<210> 194

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 194

tcctacgtac tgctgctcgt gctata 26

<210> 195

<211> 29

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 195

tgctaatact atatctacgt ctctctggg 29

<210> 196

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 196

actaacgtag gtactactgt 20

<210> 197

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 197

tactctaact actctagcgc 20

<210> 198

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 198

aaagaacaaa ctctaactag tactgg 26

<210> 199

<211> 34

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 199

ctctaggctc taacgtctaa gtaacaacag gtgg 34

<210> 200

<211> 34

<212> DNA

<213>artificial sequence

<220>

<223>TCRg fluorescence probe

<400> 200

tgtcacaggt aagttacgct acttaacaac taaa 34

<210> 201

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a fluorescence probe

<400> 201

actagtatca cctaagtact 20

<210> 202

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a fluorescence probe

<400> 202

ttaactgtag tgaatctatg tcg 23

<210> 203

<211> 30

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a fluorescence probe

<400> 203

aggtctctat gtatatccta actaggtgcg 30

<210> 204

<211> 32

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a fluorescence probe

<400> 204

ggcgtgtctc tgtgctagct agtaagtaac tc 32

<210> 205

<211> 33

<212> DNA

<213>artificial sequence

<220>

<223>TCRd/a fluorescence probe

<400> 205

taaccctcag tcgtacaaaa ggtacactct ggt 33

<210> 206

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 206

aagtacaaac gtgtaactat gt 22

<210> 207

<211> 19

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 207

tctaggtgta actatgtgg 19

<210> 208

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 208

aagctcaagc ctgctctctc acagaag 27

<210> 209

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 209

ggaacggaac gggctgctgg aaaa 24

<210> 210

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 210

aagctctggc tagaagcgtc actgaag 27

<210> 211

<211> 27

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 211

gctcgtaaat ggtgtaggct ctcctct 27

<210> 212

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 212

gctcaatggc ttaggctgct gcgt 24

<210> 213

<211> 24

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 213

tactcctgaa caacggcgca ggct 24

<210> 214

<211> 26

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 214

aagctctagc gtctagcgtc tgggta 26

<210> 215

<211> 23

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 215

agttaaatgc ggctgcctgg cgg 23

<210> 216

<211> 21

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 216

cggctggcgc gtctcagtcg t 21

<210> 217

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 217

cagtacaacg gcgcagcctc cg 22

<210> 218

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 218

aacgtggtct caatcgggcg cg 22

<210> 219

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>TCRb fluorescence probe

<400> 219

cggcctgctg cgtcacggtc 20

<210> 220

<211> 22

<212> DNA

<213>artificial sequence

<220>

<223>1 fluorescence probe of Tal

<400> 220

tacggtggct taaacaactg ct 22

<210> 221

<211> 20

<212> DNA

<213>artificial sequence

<220>

<223>1 fluorescence probe of Tal

<400> 221

acaaactacc tcgcaaccgc 20

<210> 222

<211> 13

<212> DNA

<213>artificial sequence

<220>

The patient-specific primer of<223>the 2nd subjects

<400> 1

ccgtggtgtc aca 13

Claims

Wherein the modeling module includes:

A1) sample unit, it includes the leukaemia cell's suspension for being originated from the subject, leukaemia cell in the suspension Density be 2x10⁷/ ml to 5x10⁷/ml；

A2) radiating element, it includes radioactivity processing units, for carrying out at radioactivity to immune-deficient mice appropriate Reason；

A3 it) is inoculated with unit, is used for leukaemia cell's inoculation of suspension liquid is immune scarce to handling through the radiating element In swaged mouse；

Wherein at least one described detection module includes:

It further include c) drug candidate screening module, the drug candidate screening module 2. system according to claim 1 Including the first drug candidate group and the second drug candidate group；

Wherein the first drug candidate group includes one or more drugs selected from the group below: vincristine, dexamethasone, left-handed Asparaginase, purine nucleosides analog derivative, proteasome inhibitor, mTOR inhibitors, is replaced up to sand topoisomerase enzyme inhibitor Buddhist nun, antibody class drug, all-trans retinoic acid, DNA damage inducer, folic acid reductase inhibitor, deacetylate inhibitor, mostly by Body tyrosine kinase inhibitor, cytotoxic drug and retinamide；And

Wherein the second drug candidate group includes one or more drugs selected from the group below: kinases inhibitor, cell wither Die regulator, DNA distintegrant, cell division inhibitor, MDM2 class oncogene inhibitor, antibody class drug and other chemotherapeutics Object additives.

3. system described in any one of -2 according to claim 1, wherein the modeling module further includes a4) separative unit, institute Stating separative unit includes monocyte separation medium, for being centrifugated the leukaemia cell of the subject.

4. system according to any one of claim 1-3, wherein the modeling module further includes a5) T cell removal list Member is used to remove the T cell being originated from the sample of non-T cell type leukaemia subject, to obtain the leukaemia cell Suspension.

5. system described in any one of -4 according to claim 1, wherein the target gene include it is selected from the group below a kind of or Several genes, its segment or its variant: IgH, IgK, T cell receptor A, T cell receptor D, T cell receptor B, T cell receptor G and Tal 1。

6. system according to any one of claims 1-5, wherein the detection module further includes identification unit, the identification Unit includes the reagent and device for identifying the target specificity amplified production, is expanded to identify the target specificity Increase production the characteristic amplified production that the leukaemia cell of the subject is originated from object.

7. system according to claim 1 to 6, wherein the detection module further includes analytical unit, the analysis Unit includes the reagent and device for analyzing the characteristic amplified production, to obtain the white blood for being specific to the subject The characteristic gene sequence of sick cell.

8. system described in any one of -7 according to claim 1, wherein the detection module further includes recognition unit, the identification Unit includes the reagent and device for capableing of the characteristic gene sequence of specific recognition and/or the amplification subject, from And judge the presence and/or ratio of residual leukemic cell in the subject.

9. system according to claim 1 to 8 comprising 2 or 2 or more detection modules, wherein at least One detection module is used to before the subject receives treatment detect the presence and/or ratio of wherein leukaemia cell, and Wherein at least one detection module for detected after the subject receives the treatment the wherein presence of leukaemia cell and/ Or ratio.

10. system according to claim 1 to 9, wherein the detection module includes treatment prompt unit, The successive treatment side used to corresponding subject is prompted according to the presence of leukaemia cell in subject detected and/or ratio Case.