CN111020029B - Method, primer and kit for detecting PD-L1 gene expression quantity by real-time fluorescence quantitative PCR - Google Patents

Abstract

The application discloses a method, a primer and a kit for detecting the expression quantity of a PD-L1 gene by real-time fluorescence quantitative PCR, which comprises the following steps: (1) synchronously extracting DNA and mRNA in a sample; (2) Using mRNA to be detected as a template, and synthesizing first-strand cDNA by a special reverse transcription primer; (3) The application synchronously corrects various experimental operation errors, and ensures the reliability and repeatability of the result.

Description

Method, primer and kit for detecting PD-L1 gene expression quantity by real-time fluorescence quantitative PCR

Technical Field

The application relates to a method for detecting PD-L1 gene expression quantity.

Background

Programmed cell death factor ligand 1 (PD-L1), also known as B7-H1, CD274, is an inhibitory co-stimulatory molecule encoded by the CD274 gene. PD-L1 belongs to type I transmembrane glycoprotein, has 290 amino acids and comprises an extracellular region, a hydrophobic transmembrane region and a tail cytoplasmic region. PD-L1 protein is widely expressed in lymphocytes such as activated T and B cells, macrophages, dendritic cells, thymus endothelial cells and other cells, heart, placenta and other parts, and is also highly expressed in various tumor cells and immune cells in tumor microenvironment such as lung cancer, esophageal cancer, breast cancer, lymphoma and the like, and the PD-L1 is disclosed in the literature: dong, H.et. (2002) NatMed 8,793-800, hompson, R.H.et. (2006) Cancer Res 66,3381-5 and Pardoll, D.M. (2012) Nat Rev Cancer 12,252-64 are reported in detail.

Tumor cell immune escape is one of the important reasons for tumor evasion of immune system clearance. Binding of PD-L1 expressed on the surface of tumor cells to PD-1 inhibits activation of T cells, rendering them unable to find tumors. anti-PD-L1 monoclonal antibody can effectively bind to PD-L1 on the surface of tumor cells, block signal paths, activate T cells and kill tumor cells. Blocking the PD-L1/PD-1 pathway can enhance T cell responses.

The research shows that the expression level of PD-L1 in non-myogenic invasive bladder cancer cells is related to pathological stage of tumor, and the therapeutic effect can be improved by selecting the medicament according to the expression of PD-L1. Down-regulating the mRNA expression of PD-L1 of lung adenocarcinoma A549 cells can enhance the killing effect of the killer cells on the A549 cells. The PD-L1 protein is an important index for predicting tumor postoperative progression in non-small cell lung cancer. The prognostic index effect of PD-L1 has also been reported in the study of other tumors. A large number of studies indicate that PD-L1 can play an important role as an important molecular marker in targeted therapy and prognosis diagnosis in various tumors.

The antibody medicine is prepared by using cell engineering technology and genetic engineering technology as main body, and has the advantages of high specificity, clear mechanism, obvious curative effect, less toxic side effect and the like, and has very broad prospect in treating various diseases, especially tumor. Currently, the U.S. FDA has approved the use of the anti-PD-L1 monoclonal antibody Atezolizumab (Tecentriq) in Roche/Genntech for the clinical treatment of bladder cancer and non-small cell lung cancer.

Currently, immunohistochemistry (IHC) experiments are generally used for detecting the expression status of PD-L1 in tumor cells in tumor tissues clinically, the core of the experimental method is monoclonal antibodies specifically binding to PD-L1, the performance of which directly determines the sensitivity and specificity of the whole detection, and the subjectivity of naked eye judgment is unavoidable. Therefore, the development of a novel detection method with high sensitivity and specificity for PD-L1 protein expression has important significance.

In the traditional RNA expression level detection method, mRNA such as GAPDH, beta-actin, tubulin and the like is usually used as a reference, but the expression level of the reference genes at different tissue parts is different, and the expression caused by different growth states of cells is unstable, and meanwhile, through experimental verification, the standard cannot completely and accurately reflect the condition of the expression level of the genes to be detected.

Disclosure of Invention

The application aims to provide a method for detecting the expression quantity of a PD-L1 gene by real-time fluorescence quantitative PCR and a primer used by the method, so as to overcome the defects in the prior art.

The application firstly relates to a reverse transcription primer gene sequence taking PD-L1mRNA to be detected as a template, wherein the sequence is SEQ ID NO. 1, and the code is PD-L1RT;

SEQ ID NO:1：

5’-CAATGGACTGTGTCAGTGTTGTAGCCCAGGGTCCACTGTGGGGGCTCCTCCAAATGTGTATCA-3’

the reverse transcription primer gene sequence comprises a PD-L1mRNA complementary sequence (underlined sequence), and a section of probe sequence (shaded sequence) for detecting the PD-L1mRNA reverse transcription product and a reverse primer sequence (underlined sequence) for detecting the PD-L1mRNA reverse transcription product are sequentially connected to the 5' end of the reverse transcription primer gene sequence. The primer design can ensure that the reverse transcription product can be detected by using a high-efficiency fluorescent quantitative PCR system when the length of the reverse transcription product is short or the secondary structure is complex, and the PD-L1 gene expression quantity can be detected by using a real-time fluorescent quantitative PCR technology. The amplification efficiency is optimized by optimizing the PCR reaction system and conditions.

The application also relates to a group of specific primer gene sequences taking PD-L1 reverse transcription cDNA as a template:

SEQ ID NO. 2, code: PD-L1F:5'-AGCTGAATTGGTCATCCCAGAA-3'

SEQ ID NO. 3, code: PD-L1R:5'-CAATGGACTGTGTCAGTGTTGT-3'

The application also relates to a specific probe using PD-L1 reverse transcription cDNA as a template:

SEQ ID NO. 4, code: PD-L1P:

5’-FAM-AGCCCAGGGTCCACTGTGGGGG-TAMRA-3’

the application also relates to a group of specific primer gene sequences of the reference gene RPPH 1:

SEQ ID NO. 5, code: RPPH1F:5'-CGTTCTCTGGGAACTCACCT-3'

SEQ ID NO. 6, code: RPPH1R:5'-TCCTGTCACTCCACTCCCAT-3'

The application also relates to a set of specific probes for the reference gene RPPH 1:

SEQ ID NO. 7, code: RPPH1P:

5’-HEX-AGCCCTGTTAGGGCCGCCTCTGG-TAMRA-3’

the reference gene RPPH1 is Ribonuclease P RNA Component H1, and the reference gene is as follows: j Forensic Sci.2009Mar;54 305-19, the present application is not described in detail;

the application also relates to a gene sequence of a standard substance, SEQ ID NO. 8;

the application also relates to a PCR cloning vector formed by connecting the reverse transcription product cDNA with the reference gene RPPH1 in equal proportion, which is used as a standard substance of the reference gene and the target gene to be detected: connecting the reference gene with cDNA fragments of the mRNA reverse transcription product to be detected in equal proportion, cloning the cDNA fragments to the same plasmid vector, constructing a standard product which can be used for amplification detection of the reference gene and the cDNA fragments of the mRNA reverse transcription product to be detected at the same time, diluting the obtained standard product according to concentration gradient, and drawing standard curves of the reference gene and the target gene to be detected, wherein the equal proportion is 1:1;

the cloning vector is pMD 18-TVector (Takara), which is described in the literature Wu GW, tang M, et al epitope structure of Citrus tristeza virus coatprotein mapped by recombinantprotein sand monoclone antibodies, virology 2014Jan 5;448:238-46.

The method for detecting the PD-L1 gene expression quantity by real-time fluorescence quantitative PCR comprises the following steps:

(1) Reverse transcription reaction: the method for extracting RNA/DNA in a sample is conventional, and can be seen in the specification of AllPrep DNA/RNAMini Kit of QIAGEN company, mRNA fragments to be detected are taken as templates, and a first strand cDNA is synthesized by carrying out extension reaction from the 3 'end to the 5' end of the templates under the action of reverse transcriptase through the reverse transcription primer gene sequence SEQ ID NO:1 (PD-L1 RT), wherein the reverse transcription primer is a DNA single chain with thermal stability and consists of three parts: i) The 5' end specific sequence is consistent with the forward primer sequence of QPCR and consists of 22 bases; ii) a 3' end reverse transcription primer sequence capable of specifically binding to a substrate mRNA, the sequence comprising two exon sequences spanning introns, consisting of 19 bases; iii) A specific probe sequence, which is positioned between the two DNA sequences and is formed by 22 bases as a probe corresponding sequence in QPCR;

the reaction system comprises: dNTP mix, 5*PrimeScript II Buffer (5 x reverse transcriptase buffer), primerScript II RTase (reverse transcriptase), RNase Inhibitor, RNasefile dH ₂ O, the target gene reverse transcription primer PD-L1RT is shown as SEQ ID NO. 1.

The reverse transcription reaction specifically comprises the following operation processes:

the following mixture was prepared in an RNA-free Microtube:

TABLE 1 reverse transcription of cDNA step 1

Placing in a water bath at 65 ℃ for 5 minutes, and quenching on ice.

The following reaction solution was prepared in the above mixture to a total volume of 20uL to obtain a sample DNA/RNA/Primer mixture.

TABLE 2 reverse transcription of cDNA step 2

Mix gently and place in a water bath at 45℃for 60 minutes.

Placing in a water bath at 70 ℃ for 15 minutes, and cooling on ice.

Obtaining cDNA/DNA solution for the next fluorescent quantitative PCR reaction to obtain PD-L1 reverse transcription product cDNA;

the mRNA to be detected is mRNA produced by transcription of the PD-L1 gene and is derived from sample extraction;

(2) Real-time fluorescent quantitative PCR amplification reaction: respectively adopting specific primers SEQ ID NO. 2 and SEQ ID NO. 3 corresponding to cDNA and specific primers SEQ ID NO. 4 and SEQ ID NO. 5 corresponding to reference genes as amplification primers, adopting SEQ ID NO. 4 and SEQ ID NO. 7 as specific probes, adopting the reverse transcription product cDNA and the reference gene DNA of the step (1) as templates, amplifying by double-channel real-time fluorescence quantitative PCR under the action of DNA polymerase, amplifying the target gene and the reference gene simultaneously, obtaining respective copy numbers according to respective standard curves, and calculating the copy number ratio of the target gene to the reference gene of a sample to be detected to obtain the expression level of the target gene;

the reference gene DNA is RPPH1;

the polymerase is Taq Hot Start DNA;

the reaction system comprises: dNTP mix, taq Hot Start DNA polymerase, 10 Xbuffer (Mg ²⁺ )；

The forward primer PD-L1F of the target gene is shown as SEQ ID NO. 2, the reverse primer PD-L1R of the target gene is shown as SEQ ID NO. 3, and the probe sequence PD-L1P of the target gene is shown as SEQ ID NO. 4;

reference gene forward primer RPPH1F is shown as SEQ ID NO. 5, reference gene reverse primer RPPH1R is shown as SEQ ID NO. 6, and reference gene probe sequence RPPH1P is shown as SEQ ID NO. 7;

specifically, the operation process of the real-time fluorescent quantitative PCR amplification reaction is as follows:

the mixed solution (25 uL/tube) was prepared in the following reaction system

TABLE 3 fluorescent quantitative PCR reaction System

Fluorescent quantitative PCR reaction:

TABLE 4 fluorescent quantitative PCR reaction conditions

Preferably, the real-time fluorescent quantitative PCR amplification reaction is a double-channel fluorescent quantitative PCR amplification reaction;

the term "dual channel" refers to a method of quantifying two genes simultaneously through two fluorescent channels of FAM and HEX in the same tube reaction system;

because the traditional RNA expression quantity detection method can not completely and accurately reflect the condition of the expression quantity of the gene to be detected, the application uses the genome DNA as the reference gene, and has the beneficial effects compared with the prior art that:

the application adopts a real-time fluorescence quantitative PCR technology, and an automatic instrument collects fluorescence signals, so that subjectivity of visual judgment of an IHC method is avoided, and sensitivity is improved.

The application uses the reference genome DNA as the basis of sample cell quantification to carry out homogenization treatment on the cDNA expression quantity of PD-L1, and finally determines the expression level of PD-L1 gene in the sample.

The application uses the unique standard to calculate the result after PCR, and eliminates the instability generated by taking the housekeeping gene of the self as a reference in each experiment.

The real-time fluorescent quantitative PCR is a closed detection reaction, post-treatment is not needed, the possibility of pollution is avoided, and the reliability and the repeatability of the result are ensured.

The application adopts a double-channel fluorescent quantitative PCR technology, so that the target gene and the reference gene are amplified in the same tube, all reaction conditions are uniform, and the result is more reliable.

The real-time fluorescence quantitative PCR not only realizes the leap from qualitative to quantitative of the conventional PCR, but also compared with the conventional PCR, the real-time fluorescence quantitative PCR technology collects fluorescence signals by an automatic instrument, avoids subjectivity of naked eye judgment and further improves sensitivity. The real-time fluorescent quantitative PCR is a closed detection reaction, post-treatment is not needed, the possibility of pollution is avoided, and the reliability and the repeatability of the result are ensured.

Drawings

FIG. 1 is a schematic representation of cDNA obtained by the PD-L1 reverse transcription reaction method of example 2.

FIG. 2 is a fluorescent quantitative PCR amplification method for PD-L1cDNA of example 3.

FIG. 3 is a graph of real-time fluorescent quantitative PCR amplification for a standard.

FIG. 4 is a graph of the efficiency of a plasmid standard real-time fluorescent quantitative PCR standard curve.

FIGS. 5-11 show the results of real-time fluorescent quantitative PCR amplification of samples.

FIG. 5 is a graph showing amplification of two channels PD-L1cDNA and RPPH1 obtained from MDA-MB-231 cells treated with gamma interferon according to an embodiment of the present application, FIG. 6 is a graph showing amplification of two channels PD-L1cDNA and RPPH1 obtained from K562 cells according to an embodiment of the present application, and FIGS. 7, 8, 9, 10 and 11 are graphs showing amplification of two channels PD-L1cDNA and RPPH1 obtained from tumor tissues 1, 2, 3, 4 and 5 according to an embodiment of the present application.

FIG. 12 shows the results of MDA-MB-231 cell flow cytometry.

FIGS. 13, 14, 15, 16 and 17 show the results of immunohistochemical detection of tumor tissue 1, 2, 3, 4 and 5 sections.

Detailed Description

The technical solutions in the embodiments of the present application are clearly and completely described below in conjunction with the embodiments of the present application. All other embodiments, which can be made by those skilled in the art based on the embodiments of the application without making any inventive effort, are intended to be within the scope of the application.

The reagents used in the application are as follows: allPrep DNA/RNAMini Kit from QIAGEN, DNA/RNA extraction Kit, cat# 80204; primeScript of Takara Corp ^TM II Reverse Transcriptase, reverse transcriptase, cat# 2690C; recombinant RNase Inhibitor from Takara, RNAse inhibitor, cat# 2313Q; dNTPMixture, dNTP mixture from Takara, cat# 4030 (2.5 mM), cat# 4019 (10 mM); taKaRaTaq, takara Corp ^TM Hot Start Version, cat# R007Z.

EXAMPLE 1 RNA/DNA extraction from gamma interferon treated MDA-MB-231 cells, K562 cells and tumor tissue samples

The AllPrep DNA/RNAMini Kit is used, and the DNA/RNA extraction Kit can extract 1 x 10 at most at a time ⁷ Cells or 30mg tissue.

And (3) cells: the cell number is less than or equal to 1 multiplied by 10 ⁷ 600. Mu.L Buffer RLTPlus was added; tissue: tissue was 30mg or less, and 600. Mu.L Buffer RLTPlus was added. The lysate was then centrifuged at maximum speed for 3 minutes and the upper suspension was carefully removed using a pipette.

Transfer to an Allprep DNA adsorption column (placed in a 2mL collection tube), and centrifuge at 10,000rpm for 30 seconds.

Extracting RNA: adding 70% ethanol with equal volume into the liquid centrifuged by the Allprep DNA adsorption column, and mixing.

Transfer 700uL to RNeasy column (placed in 2mL collection tube), centrifuge at 10,000rpm for 15 seconds, and pour the waste liquid from the collection tube. Repeat until all RNA was collected.

700uLBuffer RW1 was added and centrifuged at 10,000rpm for 15 seconds to pour out the waste liquid from the collection tube.

Add 500uLBuffer RPE,10,000rpm and centrifuge for 15 seconds, pour out waste liquid in the collection tube.

Add 500uLBuffer RPE,10,000rpm and centrifuge for 2 minutes, pour out waste liquid in the collection tube.

A new collection tube (1.5 mL with cap) was replaced, and 50. Mu.L of RNase-free water was added thereto, and the mixture was centrifuged at 10,000rpm for 1 minute to obtain RNA.

Extracting DNA: a new 2mL collection tube was replaced outside the Allprep DNA adsorption column.

500. Mu.L of BufferAW1 was added to the column, centrifuged at 10,000rpm for 15 seconds, and the waste liquid in the collection tube was discarded.

500. Mu.L of BufferAW2 was added to the column and centrifuged at 15,000rpm for 2 minutes.

A new collection tube (1.5 mL capped) was replaced, 50 μLBuffer EB was added and left at room temperature for 1 minute. The DNA was obtained by centrifugation at 10,000rpm for 1 minute.

Mixing the collected RNA and DNA gently for later use.

EXAMPLE 2 reverse transcription of extracted sample RNA/DNA

The following mixture was prepared in an RNA-free Microtube:

TABLE 1cDNA reverse transcription System 1

Placing in a water bath at 65 ℃ for 5 minutes, and quenching on ice.

The reaction solution was placed in the above-mentioned tube to a total volume of 20uL, and the reverse transcription reaction was performed.

TABLE 2 cDNA reverse transcription System 2

Mix gently and place in a water bath at 45℃for 60 minutes.

Placing in a water bath at 70 ℃ for 15 minutes, and cooling on ice.

A cDNA/DNA solution sample was obtained for the next fluorescent quantitative PCR reaction.

FIG. 1 is a schematic representation of a reverse transcription reaction process, as can be seen from FIG. 1: the RNA is used as a template, the PD-L1RT is used as a specific primer, and cDNA is obtained through reaction under the action of reverse transcriptase.

Example 3 Dual channel fluorescent quantitative PCR reaction and calculation of results for cDNA/DNA samples

The mixed solution (25 uL/tube) was prepared in the following reaction system

TABLE 3 fluorescent quantitative PCR reaction System

Fluorescent quantitative PCR reaction:

TABLE 4 fluorescent quantitative PCR reaction conditions

FIG. 2 shows the amplification method of PD-L1cDNA fluorescent quantitative PCR, as can be seen from FIG. 2: during PCR amplification, the 5'-3' exonuclease activity of Taq enzyme is used for carrying out enzyme digestion and degradation on the probe to separate a fluorescence emission group from a fluorescence quenching group, so that a fluorescence monitoring system can receive a fluorescence signal, namely, one fluorescence molecule is formed for each amplified DNA chain, and the accumulation of the fluorescence signal and the formation of a PCR product are completely synchronous. Thereby realizing the quantification of the PD-L1 cDNA.

Fluorescent quantitative PCR amplification results:

FIG. 3 is an amplification curve of the plasmid standard in this example.

FIGS. 5, 6 and 7 show amplification curves of the PD-L1cDNA and RPPH1 gene of the sample in this example. FIG. 3 shows the amplification curves of PD-L1cDNA and RPPH1 gene of MDA-MB-231 cells treated with gamma interferon under double fluorescent channels, wherein Ct values of the two are respectively (PD-L1 cDNA 21.61; RPPH 124.42). FIG. 4 shows the PD-L1cDNA and RPPH1 gene amplification curves of K562 cells under double fluorescent channels, wherein the Ct values are (PD-L1 cDNA27.67; RPPH 127.68), respectively. FIG. 5 shows the amplification curves of PD-L1cDNA and RPPH1 gene of tumor tissue samples under double fluorescence channels, wherein the Ct values are (PD-L1 cDNA 24.20; RPPH 127.33), respectively.

Drawing a standard curve: as shown in FIG. 3, a plasmid standard containing equal proportions of target gene cDNA and reference gene fragments is used as a template, and after two-channel fluorescent quantitative PCR detection is performed to synchronously obtain amplification curves of PD-L1cDNA and RPPH1 genes, a standard curve of the corresponding genes can be further drawn. FIG. 4 is a graph showing the standard curves of PD-L1cDNA and RPPH1 according to FIG. 3, wherein the black square curve represents the standard curve of PD-L1cDNA and the white square curve represents the standard curve of RPPH1 gene.

Assessment of PD-L1 expression level in a sample using a relative quantitative determination of gene copy number

According to the drawn standard curve, the PD-L1cDNA and RPPH1 gene copy numbers can be obtained respectively, and further, the relative quantification of the PD-L1 expression level is realized by calculating the ratio relation between the PD-L1cDNA and the RPPH1 gene copy numbers, and the specific detection and analysis results are shown in tables 5, 6 and 7.

TABLE 5 original copy number of plasmid Standard and data of fluorescent quantitative PCR results in examples

TABLE 6 fluorescent quantitative PCR result data for each sample in examples

TABLE 7 calculation results of PD-L1 Gene expression for each sample in examples

Sample detection result analysis:

in the embodiment, plasmid standard is used as a control standard, and the expression level of PD-L1 genes of MDA-MB-231 cells, K562 cells and tumor tissue samples 1-5 after gamma interferon treatment is detected.

As can be seen from Table 7, the overexpression of PD-L1 in the gamma interferon treated MDA-MB-231 cell samples is evident, consistent with the report in the literature Taube, J.M.et al. (2012) Sci Transl Med 4,127ra 37; k562 cells did not see PD-L1 expression over-expression; the PD-L1 over-expression in the tumor tissue sample 1 is obvious; no significant overexpression of PD-L1 was found in any of the tumor tissue samples 2-5. Meanwhile, the method can accurately calculate the expression quantity value of the PD-L1, and ensures the accuracy to the greatest extent.

Comparative example 4 flow cytometer detecting PD-L1 expression level in MDA-MB-231 cell sample

Cell culture plates were used to prepare primary MDA-MB-231 cells and gamma interferon-treated MDA-MB-231 cells at a concentration of 2X 10 ⁵ Plates were washed 2 times per well with 1% BSA-PBS.

Specific antibodies (Human PD-L1/B7-H1 antibodies, MAB1561, from R & D Systems) were added.

The plate was washed 2 times with 1% BSA-PBS after 1 hour at 4 ℃.

Sheep anti-mouse FITC 1 was added: 500 fluorescent markers, and shaking thoroughly.

The plate was washed 2 times with 1% BSA-PBS after 1 hour at 4 ℃.

And (5) detecting by a flow cytometer.

Analysis of detection results: FIG. 8 shows the results of MDA-MB-231 cell flow cytometry, and the expression level of PD-L1 of the gamma interferon treated MDA-MB-231 cells is significantly higher than that of untreated original MDA-MB-231 cells, which is consistent with the experimental results of the experimental method.

Comparative example 5 Immunohistochemical (IHC) detection of PD-L1 expression level in tumor tissue samples 1-5

And (5) baking the slices, namely putting the tumor tissue sample slices into an oven at 62 ℃ to bake the slices for 2 hours.

Dewaxing, immersing in xylene for 2 times each for 15 minutes, absolute ethanol for 2 times each for 5 minutes, 90% ethanol for 3 minutes, 80% ethanol for 3 minutes, and 70% ethanol for 3 minutes.

Distilled water was washed for 5 minutes, and PBS was washed 3 times for 3 minutes each.

Antigen retrieval, immersing the slices in 0.01M citric acid buffer solution (PH 6.0), heating to boiling with high microwave fire, heating with medium fire for 5 minutes, repeating for 2 times, cooling, washing with PBS for 3 times, and 3 minutes each time.

Inactivation, 3% hydrogen peroxide 15 min at room temperature, inactivation of endogenous enzymes, 3 times with PBS wash for 3 min each.

A5% BSA blocking solution was added dropwise, and the mixture was left at room temperature for 30 minutes, followed by blotting off the excess.

Specific antibodies (Human PD-L1/B7-H1 antibodies, MAB1561, from R & D Systems) were added dropwise overnight at 4 ℃.

The mixture was put into a37℃oven for 30 minutes, washed 3 times with PBS for 3 minutes each.

Biotin-labeled goat anti-mouse IgG was added dropwise, and the mixture was left at 37 ℃ for 30 minutes, and washed 3 times with PBS for 3 minutes each.

The reagent SABC was added dropwise and left at 37℃for 30 minutes. The PBS was washed 3 times for 3 minutes each.

DAB chromogenic-DAB chromogenic droplets were added to the sections using DAB chromogenic droplets. Color development was performed at room temperature for 5 minutes.

And observing the microscopic results.

Analysis of detection results: FIGS. 13-17 show immunohistochemical results of sections of tumor tissue samples 1-5, PD-L1 in tumor tissue sample 1 was expressed as weakly positive, and PD-L1 in tumor tissue samples 2-5 were all negative, consistent with the experimental method results of the present patent.

The existing FDA approved PD-L1 detection antibodies on the market have poor consistency, and the traditional IHC detection method has the defects of complex operation, high price and long time consumption (IHC detection requires 1-2 working days) in clinical detection, and cannot be standardized. Compared with the method of the application, the sensitivity is lower, and the false positive is nonspecific.

The whole detection process of the method can be completed within 3-5 hours, and full-automatic operation can be realized; in addition, the method adopts double-channel detection, is simple and convenient to operate, and can realize simultaneous detection of 40-50 samples at a time; finally, the method is simple and convenient to operate, has good stability, and the repeatability and objectivity are far better than IHC detection.

It will be appreciated that various changes and modifications may be made by those skilled in the art after reading the foregoing description of the application, and that such equivalents are intended to fall within the scope of the application as defined in the appended claims.

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Sequence listing

<110> Shanghai Kangpani medical science and technology Co., ltd

<120> method, primer and kit for detecting PD-L1 gene expression quantity by real-time fluorescence quantitative PCR

<130> 2018.3.20

<141> 2018-10-09

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aaggaccagc tctccctggg aaatgctgca cttcagatca cagatgtgaa attgcaggat 2160

gcaggggtgt accgctgcat gatcagctat ggtggtgccg actacaagcg aattactgtg 2220

aaagtcaatg ccccatacaa caaaatcaac caaagaattt tggttgtgga tccagtcacc 2280

tctgaacatg aactgacatg tcaggctgag ggctacccca aggccgaagt catctggaca 2340

agcagtgacc atcaagtcct gagtggtaag accaccacca ccaattccaa gagagaggag 2400

aagcttttca atgtgaccag cacactgaga atcaacacaa caactaatga gattttctac 2460

tgcactttta ggagattaga tcctgaggaa aaccatacag ctgaattggt catcccagaa 2520

ctacctctgg cacatcctcc aaatgaaagg actcacttgg taattctggg agccatctta 2580

ttatgccttg gtgtagcact gacattcatc ttccgtttaa gaaaagggag aatgatggat 2640

gtgaaaaaat gtggcatcca agatacaaac tcaaagaagc aaagtgatac acatttggag 2700

gagcccccac agtggaccct gggctacaac actgacacag tccattg 2747

Claims (6)

1. The reverse transcription primer gene sequence using the PD-L1mRNA to be detected as a template is shown as SEQ ID NO. 1.

2. Use of the reverse transcription primer gene sequence according to claim 1 for preparing a kit for a real-time fluorescent quantitative PCR detection method for detecting the expression amount of PD-L1 gene.

3. The use according to claim 2, the detection method comprising the steps of:

(1) Reverse transcription reaction: synchronously extracting RNA/DNA in a sample, taking an mRNA fragment to be detected as a template, and synthesizing a first strand cDNA by performing an extension reaction from the 3 'end to the 5' end of the template under the action of reverse transcriptase through a reverse transcription primer gene sequence SEQ ID NO. 1 as claimed in claim 1;

(2) Real-time fluorescent quantitative PCR amplification reaction: respectively adopting specific primers SEQ ID NO. 2, SEQ ID NO. 3 and specific primers SEQ ID NO. 5 and SEQ ID NO. 6 corresponding to the cDNA as amplification primers, SEQ ID NO. 4 and SEQ ID NO. 7 as specific probes, carrying out real-time fluorescent quantitative PCR amplification under the action of DNA polymerase by taking the reverse transcription product cDNA and the reference gene DNA of the step (1) as templates, amplifying the target gene and the reference gene simultaneously, obtaining respective copy numbers according to respective standard curves, and calculating the copy number ratio of the target gene to the reference gene of a sample to be detected to obtain the expression level of the target gene;

the reference gene DNA is RPPH1;

the forward primer of the target gene is shown as SEQ ID NO. 2, the reverse primer of the target gene is shown as SEQ ID NO. 3, and the probe sequence PD-L1P of the target gene is shown as SEQ ID NO. 4;

the forward primer of the reference gene is shown as SEQ ID NO. 5, the reverse primer of the reference gene is shown as SEQ ID NO. 6, and the probe sequence RPPH1P of the reference gene is shown as SEQ ID NO. 7.

4. The use according to claim 3, wherein the mRNA to be tested is mRNA produced by transcription of the PD-L1 gene, and is derived from sample extraction.

5. The use according to claim 3, wherein the reverse transcription reaction system comprises: dNTP Mixture 5*PrimeScriptIIBuffer,PrimerScriptIIRTase,RNase Inhibitor,RNase free dH ₂ O。

6. The use according to any one of claims 3 to 5, wherein the real-time fluorescent quantitative PCR amplification reaction is a two-channel fluorescent quantitative PCR amplification reaction.