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CN107460239A - A kind of kit for the detection of PD L1 expressions - Google Patents

A kind of kit for the detection of PD L1 expressions Download PDF

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CN107460239A
CN107460239A CN201710603539.XA CN201710603539A CN107460239A CN 107460239 A CN107460239 A CN 107460239A CN 201710603539 A CN201710603539 A CN 201710603539A CN 107460239 A CN107460239 A CN 107460239A
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kit
expression
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张道允
巩子英
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Jiaxing Permitted Medical Inspection Co Ltd
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Abstract

The present invention relates to a kind of kit for PD L1 detection of expression, including specific reverse transcription primer, Q PCR specific primers and probe；The kit can detect the height of PD L1 expressions in tumour cell.The method has simple to operate with respect to IHC, and high sensitivity, specificity are good, the advantages of only needing 2 hours just can complete to detect, and can meet the needs of clinical quick detection, direction of medication usage.

Description

A kind of kit for the detection of PD-L1 expressions
Technical field
The present invention relates to a kind of biological technical field, more particularly to a kind of kit for the detection of PD-L1 expressions.
Background technology
Immunotherapy of tumors is the immune system by transferring body, strengthens anti-tumor immunity, so as to suppress and kill Tumour cell.Immunotherapy of tumors is most one of research direction of prospect in current cancer therapies field.
Programmed death 1 (programmed death-1, PD-1) is mainly expressed in the T lymphocytes of activation, B leaching Bar cell and Macrophage Surface.Programmed death molecule ligand 1 (ligand 1, PD-L1 of programmed death 1) is PD-1 part, belong to a member of B7 superfamilies, participate in the negativity regulation of immune response.After PD-L1 is combined with PD-1, by PD-1 Inhibition signal is transmitted, regulates and controls the function of lymphocyte.The high expression PD-L1 in kinds of tumor cells surface, by with it is tumor-infiltrated The PD-1 molecules of lymphocytic cell surface combine, and suppress the function of lymphocyte, are the important originals for causing tumour that immunologic escape occurs Therefore one.Due to cell factor, such as IL-4, IL-10, INF-a ,-β or-γ induction, PD-L1 can activate the PD- in T cell 1, and the function of T cell effector is lowered, while be that tumour cell is able to invade one of mechanism of host immune monitoring.PD- L1 expresses presentation up-regulation in many solid tumors including NSCLC.27%-57.5% NSCLC cells expression PD-L1, and And PD-L1 is present in cell membrane and/or cytoplasm.In sarcoma sample NSCLC, PD-L1 expression ratio is 69%, is far above The 27% of common NSCLC.
Target spots of the PD-L1 as immunodepressant, directly acting on PD-L1 immunization therapy can discharge to immune system Brake signal, it is set to attack tumour cell.The PD-1 antagonists that have listed include Opdivo at present, Keytruda with Tecentriq.The above-mentioned three kinds of PD-1 inhibitor of FDA approveds are used for the treatment of kinds of tumors（Including non-small cell lung cancer, black Plain knurl, head and neck scale carcinoma, bladder transitional cell carcinoma etc.）.Wherein, the PD-L1 expressions of patient's body and the medicine of PD-1 immunodepressant Obvious dependency relation be present in thing curative effect.Clinical research finds, the Overall response rate of NSCLC patient positive PD-L1 for 19%~ The Overall response rate of 23%, PD-L1 negative NSCLC patient is 9%~13%, therefore expresses PD-L1 tumour to these medicines more It is sensitive.
Non-small cell lung cancer NCCN guides in 2017 clearly indicate EGFR/ALK/ROS1 the moon for PD-L1 expression >=50% The patient of property, a line can directly select PD-1 monoclonal antibody Keytruda medications.For PD-L1 expression >=1%, the past standard platiniferous During class scheme chemotherapy or after the completion of occur progression of disease Metastatic Nsclc, it is also contemplated that Keytruda Treatment.In addition, immunodepressant can also be applied to the failure of EGFR targeted therapies, the failure of ALK targeted therapies, ROS1 targeted therapies lose Lose and two, the three lines treatment after chemotherapy failure.Melanoma NCCN guides are equally explicitly pointed out with Keytruda, Opdivo etc. PD-1 monoclonal antibody classes medicine can be as the first-line treatment scheme that can not cut off or shift melanoma.In addition, other cancer guides, Such as advanced renal cell cancer, head and neck scale carcinoma Hodgkin lymphoma, equally also refer to the treatment of immunodepressant.
The content of the invention
The purpose of the present invention：A kind of kit for PD-L1 detection of expression is provided, PD-L1 in tumour cell can be detected The height of expression.The method has simple to operate with respect to IHC, and high sensitivity, specificity are good, only need 2 hours just can be complete Into the advantages of detection, can meet the needs of clinical quick detection, direction of medication usage.
To achieve these goals, the technical scheme is that：
A kind of kit for PD-L1 detection of expression, including specific reverse transcription primer, Q-PCR specific primers and probe；
The specific reverse transcription primer includes following nucleotide sequence：
PD-L1 reverse transcriptions 1： 5-AGTGCAGCCAGGTCTAATTG-3 SEQ ID NO: 01
GAPDH reverse transcriptions 2：5’ATACGACCAAATCCGTTGACT3’ SEQ ID NO：02
The Q-PCR specific primers include following nucleotide sequence：
PD-L1F：5-CATTTGCTGAACGCATTTACTG-3
PD-L1R：5-GCATTCAATTGTCATATTGCTACC-3
GAPDHF:5’CTCTGCTCCTCCTGTTCGAC3’
GAPDHR:5’ATGGTGTCTGAGCGATGTGG3’
The probe includes following nucleotide sequence：
PD-L1taqman:5’-FAM-CAAGGACCTATATGTGGTAG-MGB-3’
GAPDHTaqman:5’CGTCGCCAGCCGA3’
A kind of kit for PD-L1 detection of expression, wherein, included by the detecting step of the kit：
Extract RNA samples；
RNA reverse transcriptions are subjected to pcr amplification reaction into after cDNA in PCR amplification system in reverse transcription system.
The reverse transcription system is as follows：
5×RT buffer 4µL
Primer final concentration 200nM
dNTP 1mM
super RT 200U
RNA 6µL
DEPC water is mended to 20 μ L
The PCR amplification system is as follows：
10XPCR Buffer are diluted to 1X
dNTP 0.2mM
Template 2uL
Each primer 2 00-400 nM
Each probe 100-400 nM
Each closing probe 200-400 nM
Hot start Taq polymerase 1U
MgCL2 2-5mM
Cumulative volume 20uL.
The reaction condition of described pcr amplification reaction is as follows：
A kind of detection method of detection kit for PD-L1 detection of expression, the detection method comprise at least following steps：
Step 1：For PD-L1 design of expression synthetic primer and detection probe；
Step 2：The extraction of sample process and RNA；
Step 3：By RNA reverse transcriptions into cDNA；
Step 4：Establish pcr amplification reaction system；
Step 5：Baseline Start values, End values and threshold line, result of determination are adjusted after analysis image.
The detection method of the above-mentioned detection kit for PD-L1 detection of expression, wherein, described RNA samples include Fresh tumor tissue or fresh pathological tissue or paraffin-embedded tissue.
The detection method of the above-mentioned detection kit for PD-L1 detection of expression, wherein, the described RNA samples of sampling This is 1 gram.
Present invention employs PD-L1 specific probes and primer, the mRNA that can detect PD-L1 in tumor tissues expresses water It is flat；The innovative method using RT-PCR, compares with house-keeping gene GAPDH, by 2- △ △ CT algorithms, obtains PD-L1's Relative expression levels, its sensitivity is improved, more than traditional IHC methods；PD-L1 expression can be detected in blood；Behaviour Make simply, detection is cheap, can be with large-scale promotion；Detection speed is fast, and detection process takes around 2 hours and can completed.
Embodiment
Embodiments of the invention further explained below.
A kind of primer, probe for PD-L1 detection of expression, including PD-L1 special primers and probe, described PD-L1 Special primer is specially with probe：
The specific reverse transcription primer includes following nucleotide sequence：
PD-L1 reverse transcriptions 1：5-AGTGCAGCCAGGTCTAATTG-3 SEQ ID NO:01
GAPDH reverse transcriptions 2：5’ ATACGACCAAATCCGTTGACT3’ SEQ ID NO：02
The Q-PCR specific primers include following nucleotide sequence：
PD-L1 reverse transcription primers (PD-L1F)：5 ‘CATTTGCTGAACGCATTTACTG3’
PD-L1 reverse transcription primers (PD-L1R)：5’ GCATTCAATTGTCATATTGCTACC3’
GAPDH detection primers (GAPDHF):5’ CTCTGCTCCTCCTGTTCGAC3’
GAPDH detection primers (GAPDHR):5’ ATGGTGTCTGAGCGATGTGG3’
The probe includes following nucleotide sequence：
PD-L1taqman:5’ -FAM-CAAGGACCTATATGTGGTAG-MGB-3’
GAPDHTaqman:5’ CGTCGCCAGCCGA3’
A kind of detection method of detection kit for PD-L1 detection of expression, this method comprise at least following steps：
Step 1：The gene order for mankind's PD-1 and the GAPDH gene announced according to COSMIC data, for PD-1 and GAPDH Gene designs primer and probe.By PD-1 and GAPDH specific primers and probe system optimization, realize highly sensitive and special Different detection.
For selected PD-1 and GAPDH sequences, multipair special primer is designed using the primer-design softwares of Premier 6.0 And probe.
Step 2：Extraction detection sample RNA, described detection sample include fresh pathological tissue, paraffin-embedded tissue and Pleural fluid.
Step 3：Prepare real-time fluorescent PCR amplification reaction system.
Step 4：The Ct values shown according to fluorescent PCR amplification instrument judge testing result：The collection of fluorescence signal is set to FAM, The collection of data is scheduled on 60 DEG C.
Step 5：Experiment is analyzed and judged after terminating.
In described step 2, in addition to as follows step by step：
Step 2.1：Take about 1 × 1 cm2 each lung cancer sample slice（About 4-5 pieces section altogether）It is placed in centrifuge tube（Provide for oneself）In, add Enter 1ml dimethylbenzene, cover tightly lid, be vortexed concussion 10 seconds.
Step 2.2：12,000 rpm（~13,400 × g）Centrifugation 2 minutes, careful inhale abandon supernatant, be careful not to suction abandon it is heavy.
Step 2.3：1ml absolute ethyl alcohols are added, the concussion that is vortexed mixes；12,000 rpm are centrifuged 2 minutes, are abandoned supernatant, are paid attention to It not inhale and abandon precipitation.
Step 2.4：Open lid, room temperature or be up to 37 °C be incubated 10 minutes, until without ethanol remain.
Step 2.5：150 μ l Buffer PKD are added, precipitation is resuspended；10 μ l Proteinase K are added, be vortexed concussion Mix.
Step 2.6：56 DEG C are incubated 15 minutes, until sample is completely dissolved；80 DEG C are incubated 15 minutes；Of short duration centrifugation, makes pipe Solution on wall is collected into ttom of pipe.
Step 2.7：320 μ l Buffer RBC are added, the concussion that is vortexed thoroughly mixes.
Step 2.8：Solution resulting in step 2.7 is all added to and has been charged into collecting pipe（Collection Tube 2 ml）Filter column（DNA Remover Column）In；10,000 rpm are centrifuged 1 minute, collect filtrate.
Step 2.9：In the filtrate that step 2.8 obtains, 720 μ l absolute ethyl alcohols are added, the concussion that is vortexed thoroughly mixes.
Step 2.10：The solution of gained in step 2.9 is all added to and has been charged into collecting pipe（Collection Tube 2 ml）Adsorption column（Spin Column RS）In；10,000 rpm are centrifuged 1 minute, are outwelled the waste liquid in collecting pipe, will be adsorbed Post is placed back in collecting pipe.
Step 2.11：500 μ l Buffer RW2 are added into adsorption column, 10,000rpm centrifugations 1 minute, outwell collecting pipe In waste liquid, adsorption column is placed back in collecting pipe.
Step 2.12：12,000 rpm are centrifuged 2 minutes, outwell waste liquid in collecting pipe；Adsorption column is placed in room temperature several minutes Thoroughly to dry.
Step 2.13：By adsorption column be placed in one it is new without RNase centrifuge tubes（Collection Tube 1.5 ml）In, 20-50 μ l RNase-Free Water are vacantly added to the middle part of adsorption column, room temperature is placed 2-5 minutes, and 10,000 Rpm is centrifuged 1 minute, collects RNA solution, prepares subsequent experimental.
In described step 2.13, described reverse transcription PCR reaction system is as follows：
5×Transcription Buffer 5uL
dNTP(10mM) 1.25uL
Primer 2 00-400 nm
Ribonuclease Inhibitor (40 U/μl) 0.7uL
MLV-Transcriptase 1uL
RNA 15uL
DEPC H2O complements to 25uL
In described step 3, described pcr amplification reaction system is as follows：
Pcr amplification reaction system is as follows：
10XPCR Buffer are diluted to 1X
dNTP 0.2mM
Template 2uL
Each primer 2 00-400 nM
Each probe 100-400 nM
Each closing probe 200-400 nM
Hot start Taq polymerase 1U
MgCL2 2-5mM
Cumulative volume 20uL.
The condition of described PCR reactions is as follows：
Real-time PCR reactions condition is as follows：
The collection of fluorescence signal is set to FAM, and the collection of data is scheduled on 60 DEG C.
Step 4：Baseline Start values, End values and threshold line, result of determination are adjusted after analysis image.
After reaction terminates, instrument automatically saves result, analyzes Start values, the End values of regulation Baseline after image（Can Voluntarily adjust, Start values can be between 3-10, and between 15-20, adjust the amplification curve of negative quality-control product makes End values Its is straight or less than threshold line）And threshold line（Threshold values, it can manually adjust to baseline）.
The determination of Ct values and △ Ct values：User determines according to actual conditions at the flex point that each reaction tube expansion curve rises, Obtain its Ct value.The difference of △ Ct=mutation hole FAM fluorescence Ct values and reference opening Ct values.Mutation hole Ct values refer to sample mutation inspection Gaging hole（Mix1~mix7）Corresponding Ct values；Reference opening Ct values refer to Ct values corresponding to sample mix8.One sample may be simultaneously Containing 2 or multiple mutation types, then the sample should have corresponding to 2 or multiple △ Ct values.
As a result interpretation is as follows：As a result interpretation is as follows：Sample PD-L1 has S types curve and △ ct values>When 15, the weak tables of PD-L1 Reach；During 10≤△ ct≤15, expressed in PD-L1；During △ ct values≤10, PD-L1 strongly expresseds.
Described RNA samples include fresh tumor tissue or fresh pathological tissue or paraffin-embedded tissue.
The described RNA samples of sampling are 1 gram or so.
In described step 5, in addition to as follows step by step：
Step 5.1：Run no template control（NTC）When, FAM signals rise without curve, illustrate the pollution-free presence of experiment, Ke Yiji Continuous analysis experimental conditions.
Step 5.2：Positive quality control product analysis is run, Ct value≤30.0 of all positive quality controls, illustrates that experimental system is normal, It can continue to analyze experimental result；Its Ct value may also can be set and be fluctuated due to the different threshold values of different instruments.
Step 5.3：Run MIX5（ref）Ct values calculate, in FAM passages, if Ct value≤42.0, can continue point Analysis；If Ct values>42.0, then illustrate that sample rna degraded is serious, being not suitable for experiment needs.
Step 5.4：The definition and requirement of effective amplification curve：Typical amplification curve has three kinds of typical periods, and early stage carries on the back Scape amplification phase, Metaphase index amplification phase（Rise）The plateau risen with evening, curve global shape are S-type.Effective amplification curve is extremely Few to need background amplification phase early stage and Metaphase index to expand, curve, can calculated curve Ct values in normal S types amplification trend；Its Remaining curve is considered as invalid curve, not calculates.
Step 5.5：Ct calculating is run in FAM passages, according to the Ct values in each site and whether there is amplification curve, is judged Corresponding site situation, concrete outcome result of determination are：
As a result interpretation is as follows：Sample PD-L1 has S types curve and △ ct values>When 15, PD-L1 weak expressions；During 10≤△ ct≤15, Expressed in PD-L1；During △ ct values≤10, PD-L1 strongly expresseds.
Described mRNA reverse transcriptions cDNA operating method, comprises the following steps：
Step 1：The μ l of 13 μ L, super RT reverse transcriptases of reverse transcription reaction mixed liquor 1 are taken, are added in sterile centrifugation tube, are mixed.
Step 2：μ L, the RNA total amounts of testing sample RNA 6 are added in 0.1~5 μ g ranges to 20 μ l.
Step 3：42 ° of 1 hours of insulation, 85 °C are incubated 5 minutes；After reaction terminates, of short duration centrifugation, cooled on ice is placed in.
Step 4：Reverse transcription product can be directly used for follow-up PCR reactions and quantitative fluorescent PCR reaction.
Embodiment 1
Apply to system detection plasmid of the present invention, experiment plasmid template（Gene containing PD-L1）, detected using above-mentioned fluorescent PCR The method of PD-1 gene expressions is as follows：
（1）The processing and extraction of plasmid：
The extraction of plasmid uses TIANGEN（HighPure Plasmid kid, DP116）Plasmid extraction kit carried Take, specific operating procedure of extracting is by kit explanation operation.Carried DNA is dissolved in Tris-HCl (10mM, pH 8.0), through ultraviolet Spectrophotometer Detection and Extraction quality, determines its concentration, then adjusts DNA concentration with Tris-HCl (10mM, pH 8.0) solution To 20ng/ul as dilution mother liquor.
According to formula CCopy concentrations=（CMass concentrationX6.02X1023）/MWDNAIt is 10 to dilute wild plasmid6Individual copy number/microlitre.
By above-mentioned gained cDNA templates, the template as real-time fluorescent PCR amplification, enter performing PCR according to following amplification system Amplification:
10XPCR Buffer are diluted to 1X
dNTP 0.2mM
Template 2uL
Each primer 2 00-400 nM
Each probe 100-400 nM
Each closing probe 200-400 nM
Hot start Taq polymerase 1U
MgCL2 2-5mM
Cumulative volume 20uL；Real-time PCR reactions condition is as follows：
The collection of fluorescence signal is set to FAM, and the collection of data is scheduled on 60 DEG C.
After experiment terminates, analyzed, judged according to the following steps：
1st, no template control is run（NTC）When, FAM signals rise without curve, illustrate the pollution-free presence of experiment, can continue to analyze Experimental conditions.
2nd, positive quality control product analysis is run, Ct value≤30.0 of all positive quality controls, illustrates that experimental system is normal, Ke Yiji Continuous analysis experimental result；Its Ct value may also can be set and be fluctuated due to the different threshold values of different instruments.
3rd, the Ct values for running GAPDH calculate, and in FAM passages, if Ct value≤42.0, can continue to analyze；If Ct values> 42.0, then illustrate that sample rna degraded is serious, being not suitable for experiment needs.
4th, the definition and requirement of effective amplification curve：Typical amplification curve has three kinds of typical periods, early stage background amplification Phase, Metaphase index amplification phase（Rise）The plateau risen with evening, curve global shape are S-type.Effective amplification curve at least needs Background amplification phase early stage and Metaphase index amplification, curve, can calculated curve Ct values in normal S types amplification trend；Remaining curve It is considered as invalid curve, not calculates.
5th, Ct calculating is run in FAM passages, according to the Ct values in each site and whether there is amplification curve, judges corresponding position Point situation, concrete outcome judge as follows：Sample PD-L1 has S types curve and △ ct values>When 15, PD-L1 weak expressions；10≤△ct When≤15, expressed in PD-L1；During △ ct values≤10, PD-L1 strongly expresseds.
Sensitivity analysis：The sample DNA for taking PD-L1 gene plasmids concentration after quantitative to be 1000 copies, carries out 10 times Then 3 concentration are detected by dilution respectively, the fluorescent PCR side that 5 μ L DNA. results show the present invention is added per secondary response The high sensitivity of method, 10 copy DNA genes can detect.
Replica test：Each reaction be separately added into DNA 1000 copy, 100 copies, be repeated 10 times carry out fluorescence PCR is expanded, and 10 Ct values differ less than 0.5 circulation.
Testing result shows, detection architecture of the invention can accurately detect plasmid PD-L1 gene expressions, detection it is sensitive Degree can reach 5-10 copies.
Embodiment 2：
Clinical sample is detected with the present invention, fetches and delivers my company clinical 160, lung cancer paraffin-embedded tissue sample to be detected, man Property 83, women 77, average age be 61 years old, the median age 58 years old.Utilize special primer of the present invention and fluorescence probe PCR bodies The PD-L1 of system's 160 clinical samples of detection gene expression is as follows：
(1) sample process and RNA extraction
A. about 1 × 1 cm2 each lung cancer sample slice is taken（About 4-5 pieces section altogether）It is placed in centrifuge tube（Provide for oneself）In, add 1 ml Dimethylbenzene, lid is covered tightly, be vortexed concussion 10 seconds.
B.12,000 rpm（~13,400 × g）Centrifugation 2 minutes, careful inhale abandon supernatant, be careful not to suction abandon it is heavy.
C. 1ml absolute ethyl alcohols are added, the concussion that is vortexed mixes.12,000 rpm are centrifuged 2 minutes, are abandoned supernatant, are careful not to inhale Abandon precipitation.
D. open lid, room temperature or be up to 37 °C and be incubated 10 minutes, until being remained without ethanol.
E. 150 μ l Buffer PKD are added, precipitation is resuspended；10 μ l Proteinase K are added, the concussion that is vortexed mixes.
F.56 DEG C incubation 15 minutes, until sample is completely dissolved.80 DEG C are incubated 15 minutes.Of short duration centrifugation, makes on tube wall Solution is collected into ttom of pipe.
G. 320 μ l Buffer RBC are added, the concussion that is vortexed thoroughly mixes.
H. solution resulting in step g is all added to and has been charged into collecting pipe（Collection Tube 2 ml）'s Filter column（DNA Remover Column）In.10,000 rpm are centrifuged 1 minute, collect filtrate.
I. in the filtrate that step h is obtained, 720 μ l absolute ethyl alcohols are added, the concussion that is vortexed thoroughly mixes.
J. the solution of gained in step i is all added to and has been charged into collecting pipe（Collection Tube 2 ml）Suction Attached column（Spin Column RS）In.10,000 rpm are centrifuged 1 minute, outwell the waste liquid in collecting pipe, adsorption column is relay Reclaim in collector.
K. 500 μ l Buffer RW2 are added into adsorption column, 10,000rpm centrifugations 1 minute, are outwelled useless in collecting pipe Liquid, adsorption column is placed back in collecting pipe.
L.12,000 rpm is centrifuged 2 minutes, outwells waste liquid in collecting pipe.Adsorption column is placed in room temperature several minutes thoroughly to dry in the air It is dry.
M. by adsorption column be placed in one it is new without RNase centrifuge tubes（Collection Tube 1.5 ml）In, to absorption The middle part of post vacantly adds 20-50 μ l RNase-Free Water, and room temperature is placed 2-5 minutes, 10,000 rpm centrifugations 1 Minute, RNA solution is collected, prepares subsequent experimental.
(2) pcr amplification reaction system is established
It will put on and state RNA and be dissolved in 0.1%DEPC water, extract quality through UV spectrophotometer measuring, determine its concentration OD260/OD280 is 1.9-2.1, and reads its content.The template for taking 0.1~5 μ g RNA A to be synthesized as its c DNA, using health CDNA is synthesized for the RNA Reverse Transcriptase kits in century, cDNA synthetic systems are as follows：
5×Transcription Buffer 5uL
dNTP(10mM) 1.25uL
Ribonuclease Inhibitor (40 U/μl)（Takara） 0.7uL
MLV-Transcriptase 1uL
RNA 15uL
DEPC H2O complements to 25uL
Reverse transcription is carried out as follows using above-mentioned reverse transcription system.
(a) the μ L of 13 μ L, super RT reverse transcriptases of reverse transcription reaction mixed liquor 1 are taken, are added in sterile centrifugation tube, are mixed.
(b) μ L, the RNA total amounts of testing sample RNA 6 are added in 0.1~5 μ g ranges to 20 μ L.
（c）42 °C of 1 hours of insulation
(d) 85 °C insulation 5 minutes after cooled on ice, obtain cDNA templates
By above-mentioned gained cDNA templates, the template as real-time fluorescent PCR amplification, enter performing PCR amplification according to following amplification system：
10XPCR Buffer are diluted to 1X
dNTP 0.2mM
Template 2uL
Each primer 2 00-400 nM
Each probe 100-400 nM
Each closing probe 200-400 nM
Hot start Taq polymerase 1U
MgCL2 2-5mM
Cumulative volume 20uL；
Real-time PCR reactions condition is as follows：
The collection of fluorescence signal is set to FAM, and the collection of data is scheduled on 60 DEG C.
After experiment terminates, analyzed, judged according to the following steps：
1st, no template control is run（NTC）When, FAM signals rise without curve, illustrate the pollution-free presence of experiment, can continue to analyze Experimental conditions.
2nd, positive quality control product analysis is run, Ct value≤30.0 of all positive quality controls, illustrates that experimental system is normal, Ke Yiji Continuous analysis experimental result；Its Ct value may also can be set and be fluctuated due to the different threshold values of different instruments.
3rd, the Ct values for running GAPDH calculate, and in FAM passages, if Ct value≤42.0, can continue to analyze；If Ct values> 42.0, then illustrate that sample rna degraded is serious, being not suitable for experiment needs.
4th, the definition and requirement of effective amplification curve：Typical amplification curve has three kinds of typical periods, early stage background amplification Phase, Metaphase index amplification phase（Rise）The plateau risen with evening, curve global shape are S-type.Effective amplification curve at least needs Background amplification phase early stage and Metaphase index amplification, curve, can calculated curve Ct values in normal S types amplification trend；Remaining curve It is considered as invalid curve, not calculates.
5th, Ct calculating is run in FAM passages, according to the Ct values in each site and whether there is amplification curve, judges corresponding position Point situation, concrete outcome result of determination are：
As a result interpretation is as follows：Sample PD-L1 has S types curve and △ ct values>When 15, PD-L1 weak expressions；During 10≤△ ct≤15, Expressed in PD-L1；During △ ct values≤10, PD-L1 strongly expresseds.
Testing result, which shows to detect, the high expression of 20 PD-L1 in 160 lung cancer samples, high expression rate is 12.5%, together When above-mentioned all samples are carried out with SABCs detection contrast, SABC testing result shows>The ratio of 50% cancer cell expression For 19 patients, show that the method detection has unusual uniformity with SABC.
In summary, present invention employs PD-L1 specific probes and primer, the mRNA of PD-L1 in tumor tissues can be detected Expression；The innovative method using RT-PCR, compares with house-keeping gene GAPDH, by 2- △ △ CT algorithms, obtains PD-L1 relative expression levels, its sensitivity is improved, more than traditional IHC methods；It can detect PD-L1's in blood Expression；Simple to operate, detection is cheap, can be with large-scale promotion；Detection speed is fast, and detection process takes around 2 hours can be complete Into.
The preferred embodiments of the present invention are the foregoing is only, are not intended to limit the scope of the invention, every utilization The equivalent structure transformation that present specification is made, or directly or indirectly with the technology neck for being attached to other Related products Domain, it is included within the scope of the present invention.

Claims (7)

1. a kind of kit for PD-L1 detection of expression, it is characterised in that the kit draws including specific reverse transcription Thing, Q-PCR specific primers and probe；
The specific reverse transcription primer includes following nucleotide sequence：
PD-L1 reverse transcriptions 1： 5-AGTGCAGCCAGGTCTAATTG-3 SEQ ID NO: 01
GAPDH reverse transcriptions 2：5’ATACGACCAAATCCGTTGACT3’ SEQ ID NO：02
The Q-PCR specific primers include following nucleotide sequence：
PD-L1F：5-CATTTGCTGAACGCATTTACTG-3
PD-L1R：5-GCATTCAATTGTCATATTGCTACC-3
GAPDHF:5’CTCTGCTCCTCCTGTTCGAC3’
GAPDHR:5’ATGGTGTCTGAGCGATGTGG3’
The probe includes following nucleotide sequence：
PD-L1taqman:5’-FAM-CAAGGACCTATATGTGGTAG-MGB-3’
GAPDHTaqman:5’CGTCGCCAGCCGA3’。
2. a kind of kit for PD-L1 detection of expression according to claim 1, it is characterised in that pass through the examination The detecting step of agent box includes：
Extract RNA samples；
RNA reverse transcriptions are subjected to pcr amplification reaction into after cDNA in PCR amplification system in reverse transcription system.
A kind of 3. kit for PD-L1 detection of expression according to claim 2, it is characterised in that the reverse transcription System is as follows：
5×RT buffer 4µL
Primer final concentration 200nM
dNTP 1mM
super RT 200U
RNA 6µL
DEPC water is mended to 20 μ L.
A kind of 4. kit for PD-L1 detection of expression according to claim 3, it is characterised in that the PCR amplifications System is as follows：
10XPCR Buffer are diluted to 1X
dNTP 0.2mM
Template 2uL
Each primer 2 00-400 nM
Each probe 100-400 nM
Each closing probe 200-400 nM
Hot start Taq polymerase 1U
MgCL2 2-5mM
Cumulative volume 20uL.
5. the detection method of a kind of detection kit for PD-L1 detection of expression, it is characterised in that the detection method is at least Comprise the following steps：
Step 1：For PD-L1 design of expression synthetic primer and detection probe；
Step 2：The extraction of sample process and RNA；
Step 3：By RNA reverse transcriptions into cDNA；
Step 4：Establish pcr amplification reaction system；
Step 5：Baseline Start values, End values and threshold line, result of determination are adjusted after analysis image.
6. a kind of detection method of detection kit for PD-L1 detection of expression according to claim 5, its feature exist In described RNA samples include fresh tumor tissue or fresh pathological tissue or paraffin-embedded tissue.
7. a kind of detection method of detection kit for PD-L1 detection of expression according to claim 5, its feature exist In described RNA samples are 1 gram.
CN201710603539.XA 2017-07-23 2017-07-23 A kind of kit for the detection of PD L1 expressions Pending CN107460239A (en)

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Cited By (6)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN109652504A (en) * 2018-12-27 2019-04-19 杭州迪相实业有限公司 It is a kind of while detecting excretion body memebrane protein and the method for mRNA
CN109988842A (en) * 2019-04-19 2019-07-09 上海吉玛制药技术有限公司 A kind of reagent set and application of oligonucleotide marker probe in-situ detection PDL1
CN110268071A (en) * 2019-04-26 2019-09-20 嘉兴允英医学检验有限公司 A kind of method and kit of the detection of PD-L1 expression
CN110967484A (en) * 2019-12-10 2020-04-07 苏州药明泽康生物科技有限公司 Immunohistochemical detection test piece, kit and detection method of PD-L1
CN111020029A (en) * 2018-10-10 2020-04-17 上海康派尼恩医疗科技有限公司 Method, primer and kit for detecting PD-L1 gene expression level by real-time fluorescent quantitative PCR
CN112725418A (en) * 2021-01-25 2021-04-30 深圳乐土生物科技有限公司 Method and kit for detecting expression level of PD-L1 based on free RNA

Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN106148515A (en) * 2016-06-24 2016-11-23 暨南大学 A kind of many antigen detection methods for cerebral tumor immunization therapy
WO2017072539A1 (en) * 2015-10-27 2017-05-04 Pharmassist Ltd Method for the quantification of pd-l1 expression
CN106636392A (en) * 2016-12-16 2017-05-10 广州泰诺迪生物科技有限公司 Method for detecting tumor microenvironment-associated antigen expression

Patent Citations (3)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2017072539A1 (en) * 2015-10-27 2017-05-04 Pharmassist Ltd Method for the quantification of pd-l1 expression
CN106148515A (en) * 2016-06-24 2016-11-23 暨南大学 A kind of many antigen detection methods for cerebral tumor immunization therapy
CN106636392A (en) * 2016-12-16 2017-05-10 广州泰诺迪生物科技有限公司 Method for detecting tumor microenvironment-associated antigen expression

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
甘海涛: "共抑制因子PD-L1在大肠癌中的表达及其临床意义", 《中国优秀硕士学位论文全文数据库医药卫生科技辑》 *

Cited By (10)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN111020029A (en) * 2018-10-10 2020-04-17 上海康派尼恩医疗科技有限公司 Method, primer and kit for detecting PD-L1 gene expression level by real-time fluorescent quantitative PCR
CN111020029B (en) * 2018-10-10 2023-09-29 江苏普瑞悉恩生物科技有限公司 Method, primer and kit for detecting PD-L1 gene expression quantity by real-time fluorescence quantitative PCR
CN109652504A (en) * 2018-12-27 2019-04-19 杭州迪相实业有限公司 It is a kind of while detecting excretion body memebrane protein and the method for mRNA
CN109988842A (en) * 2019-04-19 2019-07-09 上海吉玛制药技术有限公司 A kind of reagent set and application of oligonucleotide marker probe in-situ detection PDL1
CN109988842B (en) * 2019-04-19 2022-12-06 上海吉玛制药技术有限公司 Reagent group for in-situ detection of PDL1 by oligonucleotide labeled probe and application thereof
CN110268071A (en) * 2019-04-26 2019-09-20 嘉兴允英医学检验有限公司 A kind of method and kit of the detection of PD-L1 expression
WO2020215313A1 (en) * 2019-04-26 2020-10-29 嘉兴允英医学检验有限公司 Method and kit for detecting pd-l1 expression level
CN113249447A (en) * 2019-04-26 2021-08-13 嘉兴允英医学检验有限公司 Method and kit for detecting expression level of PD-L1
CN110967484A (en) * 2019-12-10 2020-04-07 苏州药明泽康生物科技有限公司 Immunohistochemical detection test piece, kit and detection method of PD-L1
CN112725418A (en) * 2021-01-25 2021-04-30 深圳乐土生物科技有限公司 Method and kit for detecting expression level of PD-L1 based on free RNA

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