CN119020475A - A leukemia MRD detection system and kit based on third-generation sequencing - Google Patents
A leukemia MRD detection system and kit based on third-generation sequencing Download PDFInfo
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Abstract
本发明涉及一种基于三代测序白血病MRD检测系统及其试剂盒。本发明的检测系统及其试剂盒采用Cas9靶向富集三代测序技术针对MRD特异性靶点进行检测，一方面降低了二代测序全基因组检测的成本，另一方面靶向测序可以对微小缺失变异位点检测达到100X的测序深度，具有良好的市场应用前景。
The present invention relates to a leukemia MRD detection system based on third-generation sequencing and a kit thereof. The detection system and kit of the present invention use Cas9 targeted enrichment third-generation sequencing technology to detect MRD-specific targets, which reduces the cost of second-generation sequencing whole genome detection on the one hand, and on the other hand, targeted sequencing can detect small deletion variant sites to a sequencing depth of 100X, and has good market application prospects.
Description
Technical Field
The invention belongs to the field of leukemia detection, and particularly relates to a leukemia MRD detection system based on three-generation sequencing and a kit thereof.
Background
Leukemia is a malignant disease of the blood system. Leukemia is clinically divided into two major categories, acute and chronic. The marrow of patients with Acute Leukemia (AL) mainly contains primary cells and early naive cells, and has rapid cell differentiation and rapid disease progression in short term, and the natural course of the disease can be from several days to several months. Chronic Leukemia (CL) cells are mostly mature naive cells, with relatively slow disease progression, and partial natural course of disease can reach years or even decades. Acute leukemia generally threatens the life of a patient in a short period of time, so it is very important to actively explore a method for monitoring the therapeutic effect and prognosis of acute leukemia to guide clinically reasonable treatment. At present, whether the leukemia achieves an ideal therapeutic effect or not is judged clinically by the following indexes, namely complete remission (complete remission, CR): the patient has no clinical discomfort such as fever, hypodynamia and the like; normal blood flow, leukemia cells free in the leukocyte classification; the immature cells in the marrow are less than or equal to 5 percent, no Auer corpuscles, yu Gong lines and megakaryons are normal, and malignant cells are not found outside marrow. After inducing remission treatment to CR, a small fraction of malignant cells remain in the patient, which are undetectable by conventional microscopy, and we call the fraction of malignant cells still present in the patient after treatment a minimal residual disease (minimalresidul disease, MRD). The minimal residual disease is the root of the recurrence and the intractable of the acute leukemia in the future, and the reduction of the minimal residual disease in the patient is also the difficult problem of cumin research and continuous craving for attack of a plurality of students so as to bring long-term disease-free survival to the patient. In the next few years, more than ten years and even decades, the improvement of leukemia efficacy may depend mainly on two advances, one is the clinical application of targeted drugs, and the other is the personalized treatment based on dynamic evaluation of patient prognosis for trace residual disease. Currently, monitoring of MRD has become part of APL and CML treatment regimens. For acute lymphoblastic leukemia (Acute lymphoblastic leukemia, ALL), a large series of multi-center clinical tests are being carried out at home and abroad, and the treatment effect is further improved by applying tiny residual disease monitoring.
Currently, there are mainly the following methods for clinical monitoring of leukemia MRD. Cell morphology detection MRD is to search and classify naive cells under a microscope based on morphological (nuclear, cytoplasmic and granulometric) characteristics of leukemia tumor cells. Manual counting of the proportion of primary cells in bone marrow smears using an optical microscope is the earliest method used to assess leukemia MRD status, judging whether morphology is remissive, and is one of the most common detection methods at present. Immunological techniques are used for MRD monitoring for multiparameter flow cytometry (multiparameter flow cytometry, MP-FCM). The multiparameter flow cytometry is a high-throughput and high-sensitivity detection technology for detecting the expression condition of the surface or intracellular antigens of hematopoietic cells by using various antibody combinations with different fluorescent markers, and further analyzing and judging whether the serial sources, differentiation degrees and phenotypes of the cells are abnormal or not, and has become an indispensable experimental diagnosis means in diagnosis typing, treatment monitoring, prognosis evaluation and treatment target screening of malignant hematopathy. At present, the most commonly used flow cytometry is 8-10 color flow cytometry, which is one of the most widely used technologies at present, can be used for 90% -99% of patients with blood system tumor, and has the sensitivity of 0.0001%. Molecular biology techniques for monitoring MRD generally refer to the quantitative dynamic changes of abnormal genes related to leukemia tumor cells detected by PCR techniques and NGS techniques, wherein the PCR techniques mainly refer to RQ-PCR and digital PCR (Droplet DIGITAL PCR); NGS techniques are mainly the detection of specific tumor-associated targeted gene mutations, the clonal identification of IG/TCR immune repertoires and MRD monitoring. The second generation sequencing can detect the change of the load of various tumor related gene mutations (NGS-panel) at one time, and simultaneously can comprehensively evaluate factors of tumor subcloning, new cloning and clonal hematopoiesis, so compared with the PCR technology of single site detection MRD, the NGS-panel has more advantages in monitoring MRD, but the sensitivity of the common second generation sequencing technology is only about 0.001%, and in recent years, the sensitivity of the NGS-MRD can reach 0.0001% by introducing a method for reducing the PCR error rate by utilizing a molecular tag (UMI).
However, these MRD molecular monitoring techniques also suffer from various drawbacks. For example, multiparameter flow cytometry is limited by the number of detectable surface antigens and the level of technicians, and it is difficult to accurately assess the level of MRD in patients who develop clonal evolution or after bone marrow transplantation is performed. Detection of Ig/TCR gene DNA rearrangements and fusion genes (RNA molecule quantification) by RQ-PCR is technically cumbersome, complex to operate and takes a long time (around two weeks), limiting its popularization. At present, the internationally accepted BIOMED-2 standard primer system for Ig/TCR rearrangement of Euro-MRD comprises 13 PCR primer tubes, and has the advantages of huge system and complex operation. In addition, 16% of patients still have false detection in the PCR detection of MRD, and TCR/IgH VDJ rearrangement of the antigen in the treatment process also changes along with the treatment, rearrangement information can not be obtained in the diagnosis of patients, and whether rearrangement is changed can not be judged later. The second generation sequencing, although the detection accuracy is high, the probability of missing MRD can be reduced, and disease recurrence can be found earlier, the length of 500-600bp can be read at one time, and the detection rate for SNV, indel and other small-range structural variations is very good, but the structural variation missing rate of more than 50bp can be as high as 70%. Second, more than 56% of the human genome is a repeat region, including centromeres, telomeres, and other repeat elements, which results in short read sequencing that does not allow for the definition of the specific location of its short sequences during assembly of the short sequences. For example, P2RY8-CRLF2 fusion, which occurs due to X-chromosome PAR1 deletion, is difficult to correctly locate the position of DNA break during assembly due to the hundreds of kb difference in the break points. In addition, the latest NCCN guidelines list IKZF1 plus CDKN2A, CDKN2B, PAX5 or PAR1 as marker mutations with a clearly poor prognosis. However, these mutations often represent deletions of DNA fragments of the same chromosome, which are difficult to quantify with DNANGS PANEL monitoring, and transcriptome sequencing can only be monitored qualitatively, and the accuracy of the monitoring is not high, and the median is only 10-2 th power, which is difficult to use for MRD monitoring. Multiple ligation dependent probe amplification (Multiplex ligation-DEPENDENT PROBE AMPLIFICATION, MLPA) assay, proposed by the MRC company in the Netherlands, can detect multiple deletions at one time, but also makes a judgment only from a qualitative point of view, and has the problem of fuzzy identification of heterozygotes and homozygotes of a part of patients. In addition, deletion of small DNA fragments of malignant gene mutations such as EBF1, BTG1, ETV6, etc. is not applicable to molecular MRD monitoring based on conventional PCR or second generation sequencing due to ambiguous breakpoint and personalized differences among different patients. therefore, whether the conventional PCR method or the second generation sequencing method is adopted, the detection capability of detecting a large number of pathogenic malignant gene variations has a bottom defect, and the dynamic change of the MRD in the treatment process is difficult to monitor by taking the detection capability as a target, so that the development of a new MRD molecular monitoring technology is not slow.
Disclosure of Invention
The application aims to solve the technical problem of providing a leukemia MRD detection system based on three-generation sequencing and a kit thereof, which reduce the cost and realize MRD gene in-situ detection without any amplification. Furthermore, the system of the present application may be used for personalized diagnosis and disease analysis in treatment of a subject. For example, the systems and methods of the application are capable of diagnosis of leukemia from a subject and concomitant detection of MRD levels during subsequent treatments.
The invention provides a leukemia MRD detection system based on three generations of sequencing, which comprises the following steps:
a) A sample module;
Wherein the sample module comprises:
al) a sample unit comprising a subject-derived leukemia cell suspension having a density of leukemia cells in the suspension of 2x10 7/ml to 5x10 7/ml;
b) At least one detection module for detecting the presence and/or proportion of leukemia cells in the subject;
wherein the at least one detection module comprises:
b1 A cleavage unit comprising an agent capable of specifically cleaving the MRD target gene to obtain an MRD target-specific enriched product;
Wherein the reagent comprises reagent R1 and reagent R2; the reagent R1 comprises crRNA and tracrRNA for each leukemia MRD detection site and Hifi Cas9 protein; the reagent R2 comprises QuickCIP enzyme and Taq polymerase.
Further, the sample module may include:
a2 A separation unit comprising a monocyte separation liquid for centrifuging leukemia cells of the subject.
A3 A T cell removal unit for removing T cells in a sample derived from a non-T cell leukemia subject, thereby obtaining the leukemia cell suspension.
A4 A DNA extraction unit for extracting DNA of leukemia cells, thereby obtaining a DNA product of the leukemia cells.
Further, the detection module further includes:
b2 A cleavage efficiency identification unit comprising reagents and means for identifying the cleavage efficiency of the crRNA and tracrRNA of the leukemia MRD test site, thereby identifying the effectiveness of the cleavage of the crRNA and tracrRNA of the leukemia MRD test site.
B3 A data analysis unit comprising means for analyzing the data analysis after cleavage of the leukemia MRD detection site-targeted characteristic enrichment product, thereby obtaining a characteristic gene sequence specific for leukemia cells of the subject.
Further, the detection modules comprise 2 or more detection modules, wherein at least one detection module is for detecting the presence and/or proportion of leukemia cells therein prior to the subject receiving the treatment, and wherein at least one detection module is for detecting the presence and/or proportion of residual leukemia cells therein after the subject receiving the treatment.
Furthermore, the detection module is used for cutting off the DNA sequence of the leukemia MRD detection site according to the principle of CRISPR-Cas9 by aiming at the subject DNA product obtained in the a 4), after using one or more of crRNA, tracRNA and Cas9 protein complex, the crRNA and the tracRNA form gRNA, then form RNP complex with cleavage effect with Cas9 protein, and the RNP complex is specifically combined at a corresponding base position, so that the sequencing result can be obtained by sequencing on machine.
1) In the detection module, the MRD gene or fragment is one or more of the following: IKZF1, CDKN2A, CDKN2B, PAX5, CRLF2, P2RY8, ERG, ETV6, BTG1, RB1, EBF1.
2) In the detection module, the crRNA sequence of the MRD gene or fragment is at least two of the upstream and downstream of the gene or fragment respectively, and the cleavage sites are different by at least 1 kb.
3) The IKZF1 comprises a sequence selected from the group consisting of: SEQ ID NO.1, SEQ ID NO.2, SEQ ID NO.3 and SEQ ID NO. 4.
4) The CDKN2A comprises a sequence selected from the group consisting of: shown in SEQ ID NO.5 and SEQ ID NO. 6.
5) The CDKN2B comprises a sequence selected from the group consisting of: shown in SEQ ID NO.7 and SEQ ID NO. 8.
6) The PAX5 comprises a sequence selected from the group consisting of: SEQ ID NO.9, SEQ ID NO.10, SEQ ID NO.11, SEQ ID NO.12, SEQ ID NO.13, SEQ ID NO. 14.
7) The CRLF2 comprises a sequence selected from the group consisting of: SEQ ID NO.15, SEQ ID NO.16, SEQ ID NO.17, SEQ ID NO. 18: shown in SEQ ID NO.19 and SEQ ID NO. 20.
8) The P2RY8 comprises a sequence selected from the group consisting of SEQ ID NO.21, SEQ ID NO.22, SEQ ID NO.23, SEQ ID NO.24, SEQ ID NO.25 and SEQ ID NO. 26.
9) The ERG comprises a sequence selected from the group consisting of: SEQ ID NO.27, SEQ ID NO.28, SEQ ID NO.29, SEQ ID NO.30, SEQ ID NO.31, SEQ ID NO. 32.
10 The ETV6 comprises a sequence selected from the group consisting of: SEQ ID No.33, SEQ ID No.34, SEQ ID No.35, SEQ ID No.36, SEQ ID No.37, SEQ ID No. 38.
11 The BTG1 comprises a sequence selected from the group consisting of: SEQ ID NO.39, SEQ ID NO.40, SEQ ID NO.41, SEQ ID NO.42, SEQ ID NO.43, and SEQ ID NO. 44.
12 The RB1 comprises a sequence selected from the group consisting of: SEQ ID No.45, SEQ ID No.46, SEQ ID No.47, SEQ ID No.48, SEQ ID No.49 and SEQ ID No. 50.
13 The EBF1 comprises a sequence selected from the group consisting of: SEQ ID No.51, SEQ ID No.52, SEQ ID No.53, SEQ ID No.54, SEQ ID No.55, and SEQ ID No. 56.
TABLE 1 CrRNA sequences in the kit
TABLE 2 cleavage efficiency identification of PCR primer sequences in units
| Name of the name | F1 | R1 | SEQ ID NO |
| PCR1 | TCTCAAAACGCGCACAACAG | TCTGGACCCATCTCTCCCAG | 57、58 |
| PCR2 | CCATTGGACTGTGAGCCTGT | GCACAGCTGAACTCCTCCAT | 59、60 |
| PCR3 | TGAGCTCTGCTTCCCAACAG | GACATCCCCACTGAAGGAGC | 61、62 |
Further, the cutting efficiency identification unit has the following features:
1) The cleavage efficiency determining unit is used for setting PCR primers at the upstream and downstream of the cleavage site and determining whether the DNA fragment can be amplified or not and determining whether the fragment is cleaved or not.
2) Taking the IKZF1 gene as an example, the IKZF1 gene locus was selected to design PCR primers comprising a sequence selected from the group consisting of: SEQ ID NO.57, SEQ ID NO.58, SEQ ID NO.59, SEQ ID NO.60, SEQ ID NO.61, SEQ ID NO. 62.
The invention also provides a leukemia MRD detection kit based on the third generation sequencing, which comprises reagents R1 and R2; wherein the reagent R1 comprises crRNA and tracrRNA for each leukemia MRD detection site and Hifi Cas9 protein; the reagent R2 comprises QuickCIP enzyme and Taq polymerase.
Further, the leukemia MRD detection site comprises one or more of IKZF1, CDKN2A, CDKN2B, PAX5, CRLF2, P2RY8, ERG, ETV6, BTG1, RB1 and EBF 1.
Further, the concentration of the crRNA and tracrRNA is 50-100uM.
Further, the reagent R1 also includes TE buffer, nuclear FREE WATER and 10X CutSmart buffer.
Further, the reagent R2 also comprises dATP.
The invention also provides a using method of the leukemia MRD detection kit based on the third generation sequencing, which comprises the following steps:
(1) Mixing crRNA and tracrRNA, and assembling into gRNA double strand;
(2) 10. 10X CutSmart buffer was diluted with nuclear FREE WATER to give 1.25. 1.25X CutSmart Buffer; diluting the Hifi Cas9 protein by adopting the 1.25X CutSmart Buffer to obtain diluted Cas9 protein;
(3) Assembling the diluted Cas9 protein and the gRNA double strand into a ribonucleoprotein complex, and then adding QuickCIP enzyme, taq polymerase and dATP for incubation;
(4) And (3) constructing a library according to ONT PromethIon instrument instructions, and obtaining a leukemia diagnosis result after sequencing analysis.
The detection system and the kit thereof can not only track the sequence used as a diagnostic marker, but also detect the clone sequence of the newly appeared cancer cells, which possibly indicates the progress or recurrence of the disease. MRD detection with high sensitivity can predict recurrence before clinical symptoms appear, evaluate treatment effect, and help doctors to take intervention treatment to patients in early stage more timely.
Advantageous effects
(1) The detection system and the kit thereof monitor the MRD level change in the treatment process of a patient by adopting a Cas9 targeted enrichment third-generation sequencing technology, are different from a multi-parameter flow cytometry, are not limited by a large number of sample cell numbers and the number of surface antigens, do not need a large amount of experimental experience of operators, and avoid subjectivity of the operators of the multi-parameter flow cytometry. In addition, the depth of the MRD is usually 10 -4 when the MRD is detected by common multi-parameter flow cytometry (3-4 colors), and the depth of the MRD can reach 10 -6 when the MRD is detected by high-flux multi-parameter flow cytometry (more than 8 colors), but the number of cells is required to reach 2×10 7～5×107, and the remission stage specimens are generally difficult to reach; the normal precursor B cells increase during bone marrow recovery, possibly resulting in a small percentage of residual leukemia cells being masked (false negative); with the advent of CAR-T cell therapies, B cell monoclonal antibodies, biTE, etc. immunotherapy, some of the commonly used MRD detection targets such as CD19, CD22, etc. can be affected by drugs. Thus, the likelihood of false positives versus false negatives is greater for multiparameter flow cytometry, and the immune marker specificity that is relied upon is inferior to both the fusion gene and Ig/TCR rearrangement. In addition, the time for sample placement, the choice of reagent materials, the technique of operation, etc. may also cause errors, which can make multi-parameter flow cytometry detection difficult.
(2) The time for detecting Ig/TCR gene rearrangement by second generation sequencing is shorter, the technology is relatively lack of maturity, the judging standard and the like are not completely unified, and the problems to be overcome are selection standard reflecting the authenticity of leukemia cells and significant clone, standardized detection method and the like. Meanwhile, due to the limitation of short read length of the second generation sequencing, the monitoring effect on the microdeletion variation in the MRD is poor. The detection system and the kit thereof can monitor the MRD specific target by adopting a Cas9 targeted enrichment third-generation sequencing technology, so that the cost of detecting the whole genome by the second-generation sequencing is reduced on one hand, and the sequencing depth of detecting the micro deletion mutation site reaches 20X by the targeted sequencing on the other hand.
(3) The MLPA technology is a molecular biological technology based on polymerase chain reaction (Polymerase Chain Reaction, PCR) for detecting copy number variation and sequence variation of genes. The micro-deletion variation in the MRD stage can be detected, but the technology is only a qualitative detection, and the obtained result has only 3 expression forms, namely deletion homozygote, heterozygote and non-deletion. For the specific base details of the deletions, it cannot be derived therefrom. Furthermore, MLPAs are unable to identify well-defined homozygotes and heterozygotes for a portion of the sample, which requires accurate sequencing for the determination. The detection system and the kit thereof adopt Cas9 targeted enrichment third generation sequencing technology to well supplement detection targets in the technology containing the MLPA.
Drawings
FIG. 1 is a schematic diagram of a detection system according to the present invention.
FIG. 2 shows the results of the cleavage efficiency evaluation unit: a is a schematic diagram of gRNA cleavage IKZF1 gene; b is a PCR electrophoresis chart.
FIG. 3 is a sequencing peak diagram of the kit of example 2.
FIG. 4 shows the sequencing depth and coverage of the example 2 kit at each MRD detection site.
Detailed Description
The application will be further illustrated with reference to specific examples. It is to be understood that these examples are illustrative of the present application and are not intended to limit the scope of the present application. Furthermore, it should be understood that various changes and modifications can be made by one skilled in the art after reading the teachings of the present application, and such equivalents are intended to fall within the scope of the application as defined in the appended claims.
In the present application, the term "Leukemia" (Leukemia) generally refers to a disease caused by massive proliferation and accumulation of Leukemia cells, usually starting in bone marrow. The leukemia may include B-cell acute lymphoblastic leukemia and/or T-cell acute lymphoblastic leukemia.
In the present application, the term "subject" generally refers to an individual having some kind of disease characterization, which may refer to symptoms of a disease, as well as to a detrimental physiological state that cannot be altered in a prophylactic case. The individual may comprise a male and/or female, typically comprising a human or non-human animal, such as a non-human mammal. In certain embodiments, the individual includes, but is not limited to, a human, dog, cat, horse, sheep, goat, pig, cow, rabbit, rat, mouse, monkey, and the like. For example, the subject may be a human patient.
In the present application, the term "sample" generally refers to a sample for the sample unit from which a leukemia cell suspension can be obtained, which can be taken directly or indirectly from a subject. In certain embodiments, the sample taken directly from the subject is not further treated, e.g., blood may be obtained from the subject's peripheral circulatory system (e.g., by a blood lancet). The sample may include, for example, blood, urine, feces, saliva, cerebrospinal fluid, sweat, and the like. Non-limiting examples of such samples include blood (or components of blood-e.g., white blood cells, red blood cells, platelets) obtained from any anatomical location (e.g., tissue, circulatory system, bone marrow) of a subject, cells obtained from any anatomical location of a subject, skin, heart, lung, kidney, exhaled breath, bone marrow, stool, semen, vaginal fluid, interstitial fluid derived from tumor tissue, breast, pancreas, cerebrospinal fluid, tissue, throat swab, biopsy, placental fluid, amniotic fluid, liver, muscle, smooth muscle, bladder, gall bladder, colon, intestine, brain, luminal fluid, sputum, pus, microbiota (micropiota), fetal manure, milk, prostate, esophagus, thyroid, serum, saliva, urine, gastric fluid and digestive fluid, tears, ocular fluid, sweat, mucus, cerumen, oil, glandular secretions, spinal fluid, hair, nail, skin cells, plasma, nasal or nasopharyngeal wash, spinal fluid, blood, lymph fluid, and/or other excretions or body tissue. In certain embodiments, the sample is a bone marrow specimen derived from a subject, e.g., comprising bone marrow cells and/or blood cells of the subject. For another example, the sample is a bone marrow sample of a leukemia patient, wherein the leukemia cells account for more than 90%.
In the present application, the term "suspension" generally refers to a cell suspension in which one or more cells are dispersed in a liquid, wherein the cells may be separated as an individual or in a pellet consisting of no more than about 50, no more than about 40, no more than about 30, no more than about 20, no more than about 10, or less cells.
In the present application, the term "leukemia cells" generally refers to immature white blood cells resulting from the pathological differentiation of hematopoietic stem cells. For example, leukemia cells generally do not function as normal cells and can also have a strong proliferative capacity.
In the present application, the term "cell density" or "density" generally refers to the number of cells contained in a unit volume in a cell suspension. In the present application, the cell suspension can be obtained by using a medium as long as the medium enables the cells to grow normally. In certain embodiments, the medium also places the cells in suspension. For example, leukemia cell suspensions can be prepared using RPMI cell culture media. For another example, DMEM medium, IMDM medium, HAMF12 medium, MEM medium, 199 cell medium, MDSS2 cell medium, and the like can be used.
Example 1
As shown in fig. 1, the present embodiment provides a third generation sequencing leukemia-based MRD detection system, including:
a) A sample module;
Wherein the sample module comprises:
al) a sample unit comprising a subject-derived leukemia cell suspension having a density of leukemia cells in the suspension of 2x10 7/ml to 5x10 7/ml;
b) At least one detection module for detecting the presence and/or proportion of leukemia cells in the subject;
wherein the at least one detection module comprises:
b1 A cleavage unit comprising an agent capable of specifically cleaving the MRD target gene to obtain an MRD target-specific enriched product;
Wherein the reagent comprises reagent R1 and reagent R2; the reagent R1 comprises crRNA and tracrRNA for each leukemia MRD detection site and Hifi Cas9 protein; the reagent R2 comprises QuickCIP enzyme and Taq polymerase.
Further, the sample module further includes:
a2 A separation unit comprising a monocyte separation liquid for centrifuging leukemia cells of the subject.
A3 A T cell removal unit for removing T cells in a sample derived from a non-T cell leukemia subject, thereby obtaining the leukemia cell suspension.
A4 A DNA extraction unit for extracting DNA of leukemia cells, thereby obtaining a DNA product of the leukemia cells.
Further, the detection module further includes:
b2 A cleavage efficiency identification unit comprising reagents and means for identifying the cleavage efficiency of the crRNA and tracrRNA of the leukemia MRD test site, thereby identifying the effectiveness of the cleavage of the crRNA and tracrRNA of the leukemia MRD test site.
B3 A data analysis unit comprising means for analyzing the data analysis after cleavage of the leukemia MRD detection site-targeted characteristic enrichment product, thereby obtaining a characteristic gene sequence specific for leukemia cells of the subject.
Example 2
The embodiment provides a leukemia MRD detection kit based on three generations of sequencing, which comprises reagents R1 and R2;
The reagent R1 comprises the following components in percentage by weight:
1.pH7.5 TE buffer；
2. 1ul 100uM crRNA and 1ul 100uM tracrRNA for each leukemia MRD detection site;
3.50ul Nuclease Free Water(NFW)；
4.10ul 10X CutSmart buffer；
5.1ul Hifi Cas9 protein.
The reagent R2 comprises the following components in percentage by weight:
1.3ul QuickCIP enzyme；
2.1ul 10mM dATP；
3.1ul Taq polymerase.
The embodiment provides a using method of a leukemia MRD detection kit based on three generations of sequencing, which comprises the following steps:
1. The crRNA and tracrRNA were resuspended in TE buffer pH7.5 to a final concentration of 100uM (2 nmol crRNA in 20 uL; 5nmol tracrRNA in 50 uL).
2. All crRNAs to be used were equimolar mixed by adding 0.75uL of each crRNAs to tube 1.
3. The gRNA duplex was assembled in tube 2: 8uL NFW, 1uL 100uM tracrRNA, 1uL of crRNA mixture.
4. The gRNA duplex was heated at 95℃for 5min and cooled on a bench after incubation.
5. 10X CutSmart buffer1:8 was diluted with NFW in tube 3 to give 1.25X CutSmart Buffer (14uL NFW+2uL10X CutSmart buffer).
6. Hifi Cas9 1:5 was diluted in tube 4 (1uL HiFi Cas9+4uL 1.25X CutSmart) with 1.25X CutSmart.
7. Assembling Ribonucleoprotein (RNP) complexes in tube 5: 23uL NFW, 2.8uL 10X CutSmart Buffer, 3uLgRNA double strand, 1.2ul 1:5 dilution Cas9.
8. Gently stirred, then incubated at room temperature for 20 minutes, after which the RNP was placed on ice.
9. 3UL 10X CutSmart Buffer, 24ul of DNA (125 ng/ul) was prepared in tube 6.
10. 3UL QuickCIP enzyme was added to tube 6.
11. Tube 6 was incubated according to the following conditions: 10min at 37℃and 2min at 80 ℃.
12. The following components were added to the tube 6: 10uL of assembled RNP complex, 1uL 10mM dATP,1uL Taq polymerase in tube 5, incubation conditions were as follows: 37 ℃ for 15 minutes, followed by 72 ℃ for 5 minutes, after which it is kept at +12℃.
13. And (3) constructing a library according to ONT PromethIon instrument instructions, and obtaining a leukemia diagnosis result after sequencing analysis.
In this example, cleavage efficiency was identified by selecting the IKZF1 gene as an example in leukemia cell lines, and 1 cleavage site gRNA1 and gRNA2 were located upstream and downstream of the IKZF1 gene, respectively, and PCR primers were designed for the gRNA1 cleavage site, the IKZF1 gene-to-gene cleavage site and the gRNA2 cleavage site (FIG. 2A). If the site is cut, the corresponding PCR product is reduced or absent, after which the PCR product is determined using DNA agarose electrophoresis. After cleavage, the amount of PCR product of the cleavage group was clearly seen to be less than that of the control group (fig. 2B), indicating that the targeting site moiety was cleaved after addition of Cas9-gRNA mixture, such that the reduction of PCR product, CRISPR-Cas9 cleavage enrichment method was effective.
Fig. 3 shows the actual detection result of the kit of the present embodiment for a certain patient. After the crRNA cleavage site in fig. 3 is identified by the above consistent cleavage efficiency, the result of the sequencing obtained by the data analysis unit is visualized, wherein the horizontal axis represents each gene site, the vertical axis represents the number of reads sequenced, and the condition of gene deletion at the MRD detection site, such as partial exon deletion in IKZF1 gene, total deletion of CDKN2A and CDKN2B genes, can be known.
FIG. 4 is the actual test results of the kit of this example for a patient, which analyzed the sequencing depth and sequencing coverage of each MRD test site, demonstrating the complete deletion of the partial exons and CDKN2A and CDKN2B genes within the IKZF1 gene.
Claims (11)
1. A three-generation sequencing-based leukemia MRD detection system, comprising:
a) A sample module;
Wherein the sample module comprises:
al) a sample unit comprising a subject-derived leukemia cell suspension having a density of leukemia cells in the suspension of 2x10 7/ml to 5x10 7/ml;
b) At least one detection module for detecting the presence and/or proportion of leukemia cells in the subject;
wherein the at least one detection module comprises:
b1 A cleavage unit comprising an agent capable of specifically cleaving the MRD target gene to obtain an MRD target-specific enriched product;
Wherein the reagent comprises reagent R1 and reagent R2; the reagent R1 comprises crRNA and tracrRNA for each leukemia MRD detection site and Hifi Cas9 protein; the reagent R2 comprises QuickCIP enzyme and Taq polymerase.
2. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the sample module further comprises a 2) a separation unit comprising a mononuclear cell separation liquid for centrifugation of leukemia cells of the subject.
3. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the sample module further comprises a 3) a T cell removal unit for removing T cells in a sample derived from a non-T cell leukemia subject, thereby obtaining the leukemia cell suspension.
4. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the sample module further comprises a 4) a DNA extraction unit for extracting DNA of leukemia cells, thereby obtaining DNA products of the leukemia cells.
5. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the detection module further comprises b 2) a cleavage efficiency identification unit comprising reagents and means for identifying the cleavage efficiency of the crRNA and tracrRNA of the leukemia MRD detection site, thereby identifying the effectiveness of the cleavage of the crRNA and tracrRNA of the leukemia MRD detection site.
6. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the detection module further comprises b 3) a data analysis unit comprising means for analyzing the leukemia MRD detection site-targeted characteristic enrichment product post-cleavage data analysis, thereby obtaining a characteristic gene sequence specific for leukemia cells of the subject.
7. The three-generation sequencing leukemia based MRD detection system of claim 1, wherein: the detection modules comprise 2 or more detection modules, wherein at least one detection module is for detecting the presence and/or proportion of leukemia cells therein prior to the subject receiving treatment, and wherein at least one detection module is for detecting the presence and/or proportion of residual leukemia cells therein after the subject receiving the treatment.
8. The utility model provides a leukemia MRD detection kit based on three generations sequencing which characterized in that: comprising reagents R1 and R2; wherein the reagent R1 comprises crRNA and tracrRNA for each leukemia MRD detection site and Hifi Cas9 protein; the reagent R2 comprises QuickCIP enzyme and Taq polymerase.
9. The test kit of claim 8, wherein: the leukemia MRD detection site comprises one or more of IKZF1, CDKN2A, CDKN, B, PAX, CRLF2, P2RY8, ERG, ETV6, BTG1, RB1 and EBF 1.
10. The test kit of claim 8, wherein: the reagent R1 also includes TE buffer, nuclear FREE WATER and 10X CutSmart buffer.
11. The application method of the leukemia MRD detection kit based on the three generations of sequencing comprises the following steps:
(1) Mixing crRNA and tracrRNA, and assembling into gRNA double strand;
(2) 10. 10X CutSmart buffer was diluted with nuclear FREE WATER to give 1.25. 1.25X CutSmart Buffer; diluting the Hifi Cas9 protein by adopting the 1.25X CutSmart Buffer to obtain diluted Cas9 protein;
(3) Assembling the diluted Cas9 protein and the gRNA double chain into a ribonucleoprotein complex, adding sample DNA for MRD detection site cutting enrichment, and then adding QuickCIP enzyme, taq polymerase and dATP for incubation;
(4) And (3) establishing a library according to ONT PromethIon instrument instructions, and obtaining a leukemia diagnosis result after data analysis by a data analysis unit.
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