CN110699435A - Kit for detecting PD-L1 expression level - Google Patents
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Abstract
The invention belongs to the technical field of PD-L1 expression detection, and particularly relates to a kit for detecting the expression level of PD-L1. The kit comprises a PD-L1 specific primer, a GAPDH specific primer, a PD-L1 probe and a GAPDH probe. Compared with the prior art, the kit provided by the invention is based on a digital PCR detection system, full-automatic detection is carried out after sample loading, manual operation interference is reduced, the result is stable, the accuracy is high, the data amplification effect is good, and the kit can be more conveniently used for auxiliary diagnosis and clinical treatment guidance.
Description
Technical Field
The invention belongs to the technical field of PD-L1 expression detection, and particularly relates to a kit for detecting the expression level of PD-L1.
Background
As the national food and drug administration (CFDA) officially approves the first PD-1 inhibitor nivolumab in China to be on the market, the new era of immunotherapy in China is indicated. The tumor immunotherapy is to enhance the immunity against tumor by mobilizing the body's own immune system, thus inhibiting and killing tumor cells, and its appearance changes the traditional pattern of tumor surgery, radiotherapy and chemotherapy, and is one of the most promising research directions in the current tumor therapy field.
In the interaction between tumor cells and immune effector cells, the combination of programmed cell death-ligand 1 (PD-L1) on the surface of tumor cells and programmed cell death-receptor 1 (PD-1) on the surface of immune effector cells (T lymphocytes) transmits immunosuppressive signals, and is an important mechanism for tumor cells to escape lymphocyte killing. The research shows that PD-1/PD-L1 is obviously up-regulated in various tumor tissues and is a widely expressed biomarker. The immunosuppressant taking PD-1 and PD-L1 as targets can block the combination of PD-1 and PD-L1, block negative regulation signals, recover the activity of T cells, and enhance the capacity of resisting tumor growth and metastasis. Therefore, the method has important clinical significance for screening cancer patients with tumor tissues or cells highly expressing PD-1/PD-L1 by using a proper detection method so as to pertinently carry out the treatment of targeted drugs.
At present, the detection of PD-1/PD-L1 is mainly based on protein level, and the clinical experiment mainly adopts an Immunohistochemical (IHC) method, namely, tumor tissues obtained after operation or puncture are subjected to section staining, and after antibody staining, the expression condition is judged according to the shade of color. Therefore, in addition to the dyeing technology and conditions, the specificity and the pathological evaluation standard of the antibody are particularly important, and the existing detection kit has the defects of low sensitivity, low specificity, poor stability and the like due to the existence of the factors, so that the detection market of PD-1/PD-L1 in China is relatively disordered and the detection value is relatively low. Therefore, a unified and quantifiable detection method is urgently needed to screen out tumor patients with high expression of PD-1/PD-L1 in clinic, and guidance and basis are provided for personalized immunotherapy.
The current realization of quantitative detection is mainly detection by real-time fluorescent quantitative PCR,
for example, patent CN107460239A discloses a kit for detecting the expression level of mRNA in tumor tissue by RT-PCR method. Compared with housekeeping gene GAPDH, the relative expression level of PD-L1 is obtained by a 2-delta CT algorithm in the kit, the operation is simple, and the detection can be completed within 2 min.
However, this detection method does not provide absolute quantification on the one hand and, in addition, has a relatively high lower limit of detection.
Disclosure of Invention
In order to solve the above problems in the prior art, the invention of the present application aims to provide a kit for detecting the expression level of PD-L1. The kit can detect the expression of the PD-L1 gene in the tumor tissue and provides absolute quantification.
In order to achieve the above object, the present invention provides the following technical solutions:
a kit for detecting the expression level of PD-L1, which is characterized by comprising a PD-L1 specific primer, a GAPDH specific primer, a PD-L1 probe and a GAPDH probe;
the PD-L1 specific primer comprises the following nucleotide sequence
Upstream primer 5-CCGCTGCATGATCAGCTATGG-3SEQ ID NO: 1;
downstream primer 5-GAGGTGACTGGATCCACAACC-3SEQ ID NO. 2;
the PD-L1 probe comprises the following nucleotide sequence
5-CAAGGACCTATATGTGGTAG-3SEQ ID NO:3；
The GAPDH specific primer comprises the following nucleotide sequence
Upstream primer 5-CTCTGCTCCTCCTGTTCGAC-3SEQ ID NO. 4;
downstream primer 5-ATGGTGTCTGAGCGATGTGG-3SEQ ID NO. 5;
the GAPDH probe comprises the following nucleotide sequence
5-CCTGGCCAAGGTCATCCATGACAACTT-3SEQ ID NO:6。
Preferably, the kit further comprises a PCR amplification premix, and the amplification premix comprises
Preferably, the fluorescence reporter group of the PD-L1 probe is CY3, and the fluorescence quencher group is BHQ 1; the fluorescent reporter group of the fluorescent probe of the GAPDH probe is FAM, and the fluorescent quencher group is BHQ 1.
The invention also provides a method for detecting the expression level of PD-L1 by using the kit, which comprises the following steps:
s1 designing PD-L1 specific primers and probes and GAPDH specific primers and probes against selected PD-L1 and GAPDH sequences;
s2 extracting RNA of the detection sample;
s3 reverse transcribing RNA into cDNA in a reverse transcription PCR reaction system;
s4 carrying out amplification reaction in the PCR amplification reaction system.
Preferably, the reverse transcription described in S3 uses Invitrogen SuperScript IV First-StrandSynthesis System (Cat. No. 18091050), and the PCR reaction System comprises the following components:
preferably, the reverse transcriptase is SuperScriptTMIV Reverse Transcriptase(200U/μL)；
The transcription buffer solution is 5X SSIV buffer solution;
the Ribonuclease Inhibitor is Ribonuclease Inhibitor (40U/. mu.L).
Preferably, the test sample is selected from fresh pathological tissue, paraffin-embedded tissue or thoracic fluid.
Preferably, the amplification reaction procedure described in S4 includes the steps of:
preferably, the reverse transcription reaction described in S3 is performed under conditions of 65 ℃ for 5min, ice for at least 1min, room temperature extension for 10min, and 70 ℃ for 5min to inactivate the enzyme.
Preferably, the detection method further comprises S5 interpretation analysis of the detection result, which comprises the following operations:
1) if at least one of the detection concentrations of the PD-L1 gene and the GAPDH reference gene is less than 10 copies/mu L, the detection result is invalid, and the sample nucleic acid is required to be extracted again for detection;
2) if the detected concentrations of the PD-L1 gene and the GAPDH reference gene are both at 1 x 102copies/μL-1╳105Between copies/. mu.L, the following calculation was performed directly:
the detection concentration of the PD-L1 gene/the detection concentration of the GAPDH reference gene,
when N is less than or equal to 0.2, the result is interpreted as: the expression level of PD-L1 is normal;
when N is more than 0.2, the result is interpreted as: high expression of PD-L1;
3) if at least one of the detected concentrations of the PD-L1 gene and the GAPDH reference gene is more than 1 x 105copies/. mu.L, the sample to be tested needs to be diluted to a linear range for re-measurement, and the result is judged according to the standard of 2).
Compared with the prior art, the kit provided by the invention has the following beneficial effects:
1) the matched PD-L1 gene expression detection kit provided by the invention is based on a digital PCR detection system, full-automatic detection is carried out after sample loading, manual operation interference is reduced, the result is stable, the accuracy is high, the data amplification effect is good, and the kit can be more conveniently used for auxiliary diagnosis and clinical treatment guidance.
Compared with the current similar detection products, the detection method has the following specific advantages: compared with real-time fluorescent quantitative PCR, the method can carry out absolute quantification, the lowest limit of the sample concentration can reach 1 ng/mu L, and the lower limit of the detection can reach 10 copies/mu L.
2) The kit uses the independently designed primer probe combination, and has high specificity and better detection effect.
3) The kit uses an autonomous optimized reaction system, greatly improves the porosity of the chip on the premise of ensuring high efficiency and accuracy of the reaction, and reduces false positive reaction by using hot-start DNA polymerase.
Detailed Description
In order to make the purpose and technical solution of the embodiments of the present invention clearer, the technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention.
Example 1
A kit for detecting the expression level of PD-L1, which comprises a PD-L1 specific primer, a GAPDH specific primer, a PD-L1 probe and a GAPDH probe;
the PD-L1 specific primer comprises the following nucleotide sequence
Upstream primer 5-CCGCTGCATGATCAGCTATGG-3SEQ ID NO: 1;
downstream primer 5-GAGGTGACTGGATCCACAACC-3SEQ ID NO. 2;
the PD-L1 probe comprises the following nucleotide sequence
5-CAAGGACCTATATGTGGTAG-3SEQ ID NO:3；
The GAPDH specific primer comprises the following nucleotide sequence
Upstream primer 5-CTCTGCTCCTCCTGTTCGAC-3SEQ ID NO. 4;
downstream primer 5-ATGGTGTCTGAGCGATGTGG-3SEQ ID NO. 5;
the GAPDH probe comprises the following nucleotide sequence
5-CCTGGCCAAGGTCATCCATGACAACTT-3SEQ ID NO:6。
A method for detecting the expression level of PD-L1 by using the kit comprises the following steps:
s1 designing PD-L1 specific primers and probes and GAPDH specific primers and probes for selected PD-L1 and GAPDH sequences using Premier 5.0 primer design software;
s2 extracting RNA of the detection sample; the detection sample is selected from fresh pathological tissues, paraffin-embedded tissues or thoracic cavity fluid; extraction of RNA Qiagen RNeasy Mini Kit (Cat. No. 74104) was used, following Kit instructions.
S3 reverse transcribing RNA into cDNA in a reverse transcription PCR reaction system;
s4 carrying out amplification reaction in the PCR amplification reaction system.
The specific operation of extracting the RNA of the detection sample in step S2 is as follows:
1) the tissue sample is not more than 30mg, 600 mu L of RLT buffer solution is added after the tissue is fully ground, the tissue is shaken and uniformly mixed, then the tissue is centrifuged at 13000rpm for 3min, and the supernatant is carefully sucked for the next step;
2) adding equal volume of 70% ethanol into the lysate, blowing, beating and mixing uniformly, and immediately carrying out the third step without centrifugation;
3) transferring sample (containing precipitate) of at most 700 μ L into adsorption column, covering, centrifuging at more than 8000g for 15s, and pouring out liquid in collection tube;
4) adding 700 μ L RW1 buffer solution into adsorption column, covering cover, centrifuging at a speed of more than 8000g for 15s, and discarding liquid in collection tube;
5) adding 500 μ L RPE buffer solution into adsorption column, covering cover, centrifuging at a speed of more than 8000g for 15s, and discarding liquid in collection tube;
6) adding 500 μ L RPE buffer solution into adsorption column, covering, and centrifuging at rotation speed of more than 8000g for 2 min;
7) placing the adsorption column in a new collection tube, adding 30-50 μ L RNase-free water into the adsorption column, covering the adsorption column, centrifuging for 1min to dissolve RNA more than 8000 g;
8) if it is desired to increase the recovery, step 7 may be repeated or the RNA solution in the collection tube may be re-adsorbed onto the adsorption column for reconstitution.
In step S3, the reverse transcription PCR reaction system includes the following components:
the reaction conditions were 65 deg.C for 5min, ice for at least 1min, room temperature extension for 10min, and 70 deg.C for 5min to inactivate the enzyme.
In step S4, the PCR amplification reaction system comprises the following components
The amplification reaction procedure described in S4 includes the following steps:
the fluorescence detection channels were FAM and CY 3.
Interpretation of the results S5
1) If at least one of the detection concentrations of the PD-L1 gene and the GAPDH reference gene is less than 10 copies/mu L, the detection result is invalid, and the sample nucleic acid is required to be extracted again for detection;
2) if the detected concentrations of the PD-L1 gene and the GAPDH reference gene are both at 1 x 102copies/μL-1╳105Between copies/. mu.L, the following calculation was performed directly:
the detection concentration of the PD-L1 gene/the detection concentration of the GAPDH reference gene,
when N is less than or equal to 0.2, the result is interpreted as: the expression level of PD-L1 is normal;
when N is more than 0.2, the result is interpreted as: high expression of PD-L1;
3) if at least one of the detected concentrations of the PD-L1 gene and the GAPDH reference gene is more than 1 x 105copies/. mu.L, the sample to be tested needs to be diluted to a linear range for re-measurement, and the result is judged according to the standard of 2).
In this example, three samples were tested, and the interpretation results were as follows:
| Samples | PD-L1 copy number | GAPDH copy number | PD-L1/GAPDH | Determination of results |
| 1 | 5632 | 29552 | 0.190579318 | Normal expression |
| 2 | 9043 | 28650 | 0.315636998 | High expression |
| 3 | 4980 | 27590 | 0.180500181 | Normal expression |
The interpretation of the results is: the high expression of PD-L1 in sample 2 of 3 samples and the normal expression of PD-L1 in the other two samples are the same as the detection result of immunohistochemistry.
Test example 1
1) The qPCR reaction was performed using primers and probes designed for PD-L1, and the reaction system was as follows:
the PCR reaction temperature cycling conditions were as follows:
taking the PCR product to carry out electrophoresis detection, wherein the electrophoresis detection result shows that only one amplification band is available, and the size of the band is consistent with that of the amplification product (100-200 bp).
2) And (2) taking a PCR product, purifying the PCR product by using a Qiagen MinElute PCR purification kit (Cat.No.28004), uniformly mixing the purified product with a pMD18-T vector (Cat.No 6011, TAKARA), connecting at 16 ℃ overnight, adding the connecting product into Ecoli JM109 competent cells, coating the competent cells on a solid LB plate containing an X-gal substrate, an IPTG inducer and Amp, culturing in a 37 ℃ constant temperature incubator overnight (12-16h), and randomly selecting 30 positive single clones for Sanger generation sequencing. The sequencing results were aligned with DNAstar, and 30 monoclonal sequencing results were completely matched with the primer design region. Thus, the designed primer and the probe have better specificity.
Test example 2
80 clinical lung cancer paraffin-embedded tissue samples are detected by using the invention, wherein 48 male patients and 32 female patients have the average age of 63 years. The gene expression of PD-L1 of 80 clinical samples detected by the specific primers and the probes of the invention matched with a digital PCR system is as follows:
(1) sample processing and RNA extraction
1. About 1 x 1cm2Placing 4-5 lung cancer sample slices in a centrifuge tube, adding 1ml xylene, covering the tube cover tightly, carrying out vortex oscillation for 10s, centrifuging at 12000rpm for 2min at room temperature, sucking out the supernatant by using a gun head, paying attention to not remove precipitates, and repeating the step for three times;
2. adding 1mL of absolute ethyl alcohol into the centrifugal tube after the sediment is discarded, shaking uniformly, centrifuging at 12000rpm for 5min at room temperature, sucking out the supernatant by using a gun head, paying attention to not remove the sediment, and repeating the step twice;
3. standing at room temperature for 5-10min, and volatilizing ethanol to obtain dry and anhydrous sample;
4. adding 600. mu.L of RLT buffer solution, shaking and mixing uniformly, and then centrifuging at 13000rpm for 3 min. Carefully aspirate the supernatant for the next step;
5. adding equal volume of 70% ethanol into the lysate, blowing, beating and mixing uniformly, and immediately carrying out the next step without centrifugation;
6. up to 700. mu.L of the sample (containing the precipitate) was transferred to the adsorption column, covered and centrifuged for 15s at > 8000g, and the liquid was decanted from the collection tube.
7. Adding 700 μ L RW1 buffer solution into adsorption column, covering the cover, centrifuging at a speed of more than 8000g for 15s, and discarding the liquid in the collection tube.
8. Adding 500 μ L RPE buffer solution into adsorption column, covering cover, centrifuging at a speed of more than 8000g for 15s, and discarding liquid in collection tube.
9. Adding 500 μ L RPE buffer solution into adsorption column, covering, and centrifuging at rotation speed of more than 8000g for 2 min;
10. placing the adsorption column in a new collection tube, adding 30-50 μ L RNase-free water into the adsorption column, covering the adsorption column, centrifuging for 1min to dissolve RNA more than 8000 g;
11. if it is desired to increase the recovery, step 7 may be repeated or the RNA solution in the collection tube may be re-adsorbed onto the adsorption column for reconstitution.
(2) Reverse transcription and PCR amplification reactions
The RNA is dissolved in DEPC water, the extraction quality is detected by an ultraviolet spectrophotometer, the concentration is determined, and the OD260/OD280 of all samples is between 1.9 and 2.1. Taking 1-5 mu g of RNA as a template for cDNA synthesis, synthesizing cDNA by adopting an Invitrogen SuperScript IV reverse transcription kit, wherein the synthesis system is as follows:
the reaction conditions were 65 deg.C for 5min, ice for at least 1min, room temperature extension for 10min, and 70 deg.C for 5min to inactivate the enzyme.
(3) Digital PCR amplification reaction
The synthesized cDNA is diluted to a certain concentration, and the reaction system is as follows:
the reaction conditions are as follows:
the fluorescence signals were CY3 and FAM.
(4) Analysis and interpretation of experimental results
1. When a template-free control (NTC) is operated, both the CY3 channel and the FAM channel have no positive points, which indicates that the experiment has no pollution and the experiment condition can be continuously analyzed.
2. When the positive quality control product is operated, the number of positive points of the CY3 and FAM channels is in an effective range, which indicates that the experimental system is normal, and the experimental result can be continuously analyzed.
3. The amount of CY3 and FAM channel positive points varies according to the sample input amount, and the amount of positive points of GAPDH under the reasonable dilution multiple of the sample is about 1 x 104copies/. mu.L, PD-L1, was varied depending on whether the sample itself was highly expressed.
4. And (3) comparing the detection results of the clinical samples:
as can be seen from the above table, the digital PCR method of the present invention has high consistency with the detection result of immunohistochemistry, and the detection result is reliable.
In conclusion, the PD-L1 specific probe and primer are adopted in the invention, so that the expression level of mRNA of PD-L1 in tumor tissues can be detected; compared with housekeeping gene GAPDH, the relative expression level of PD-L1 is obtained by absolutely quantifying two cDNAs by means of a self-developed digital PCR platform, the detection sensitivity is improved, and the expression of PD-L1 can be detected in blood; simple operation, low price and large-scale popularization.
The above are merely embodiments of the present invention, which are described in detail and with particularity, and therefore should not be construed as limiting the scope of the invention. It should be noted that, for those skilled in the art, various changes and modifications can be made without departing from the spirit of the present invention, and these changes and modifications are within the scope of the present invention.
Claims (10)
1. A kit for detecting the expression level of PD-L1, which is characterized by comprising a PD-L1 specific primer, a GAPDH specific primer, a PD-L1 probe and a GAPDH probe;
the PD-L1 specific primer comprises the following nucleotide sequence
Upstream primer 5-CCGCTGCATGATCAGCTATGG-3SEQ ID NO: 1;
downstream primer 5-GAGGTGACTGGATCCACAACC-3SEQ ID NO. 2;
the PD-L1 probe comprises the following nucleotide sequence
5-CAAGGACCTATATGTGGTAG-3SEQ ID NO:3；
The GAPDH specific primer comprises the following nucleotide sequence
Upstream primer 5-CTCTGCTCCTCCTGTTCGAC-3SEQ ID NO. 4;
downstream primer 5-ATGGTGTCTGAGCGATGTGG-3SEQ ID NO. 5;
the GAPDH probe comprises the following nucleotide sequence
5-CCTGGCCAAGGTCATCCATGACAACTT-3SEQ ID NO:6。
2. The kit for detection of the expression level of PD-L1 according to claim 1, further comprising a PCR amplification premix comprising
3. The kit for detecting the expression level of PD-L1 according to claim 1 or 2, wherein the fluorescence reporter group of the PD-L1 probe is CY3, and the fluorescence quencher group is BHQ 1; the fluorescent reporter group of the fluorescent probe of the GAPDH probe is FAM, and the fluorescent quencher group is BHQ 1.
4. A method for detecting the expression level of PD-L1 using the kit of any one of claims 1-3, comprising the steps of:
s1 designing PD-L1 specific primers and probes and GAPDH specific primers and probes against selected PD-L1 and GAPDH sequences;
s2 extracting RNA of the detection sample;
s3 reverse transcribing RNA into cDNA in a reverse transcription PCR reaction system;
s4 carrying out amplification reaction in the PCR amplification reaction system.
5. The method according to claim 4, wherein the reverse transcription reaction system in S3 comprises the following components:
6. the method of claim 5, wherein the reverse transcriptase is 200U/. mu.L SuperScriptTMIV ReverseTranscriptase；
The transcription buffer solution is 5X SSIV buffer solution;
the Ribonuclease Inhibitor is 40U/muL Ribonuclease Inhibitor.
7. The method of claim 4, wherein the test sample is selected from the group consisting of fresh pathological tissue, paraffin-embedded tissue, and thoracic fluid.
8. The method for detecting the expression level of PD-L1 according to claim 4, wherein the amplification reaction program described in S4 includes the following steps:
9. the method according to claim 4, wherein the reverse transcription reaction is performed at S3 under the conditions of 65 ℃ for 5min, ice for at least 1min, room temperature extension for 10min, and enzyme inactivation at 70 ℃ for 5 min.
10. The method according to claim 4, further comprising S5 interpretation analysis of the test results, as follows:
1) if at least one of the detection concentrations of the PD-L1 gene and the GAPDH reference gene is less than 10 copies/mu L, the detection result is invalid, and the sample nucleic acid is required to be extracted again for detection;
2) if the detected concentrations of the PD-L1 gene and the GAPDH reference gene are both at 1 x 102copies/μL-1╳105Between copies/. mu.L, the following calculation was performed directly:
the detection concentration of the PD-L1 gene/the detection concentration of the GAPDH reference gene,
when N is less than or equal to 0.2, the result is interpreted as: the expression level of PD-L1 is normal;
when N is more than 0.2, the result is interpreted as: high expression of PD-L1;
3) if at least one of the detected concentrations of the PD-L1 gene and the GAPDH reference gene is more than 1 x 105copies/. mu.L, the sample to be tested needs to be diluted to a linear range for re-measurement, and the result is judged according to the standard of 2).
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