PD-1 and PD-L1 gene polymorphism detection kit and use method thereof

Abstract

The invention discloses a PD-1 (programmed death-1) and PD-L1 (programmed death-L1) gene polymorphism detection kit and a use method thereof. The kit is used for detecting an rs34819629 site of a PD-1 gene and an rs2297137 site of a PD-L1 gene, four groups of primer sequences are designed, and the kit further comprises a PCR (Polymerase Chain Reaction) reaction buffer solution, dNTPs (Deoxyribose Nucleic Acid), DNA (Deoxyribonucleic Acid) polymerase and template DNA. The kit disclosed by the invention is an efficient tool for detecting the specific polymorphism of the PD-1 and PD-L1 genes, and has the characteristics of high accuracy, simplicity in operation and capability of supporting personalized medical treatment. Through the detection kit, targeted immunotherapy suggestions can be provided for patients, the effectiveness of immunotherapy is remarkably improved, important gene information is provided for related researches, and development of diabetes immunotherapy is promoted.

Description

Technical Field

[0001] The present invention relates to a kit, specifically a kit for detecting PD-1 and PD-L1 gene polymorphisms and a method for using the same. Background Art

[0002] In the field of diabetes immunotherapy, PD-1 (programmed death protein 1) and PD-L1 (programmed death ligand 1), as key immune checkpoints, have received extensive attention. Research shows that PD-1 is usually expressed on T cells in peripheral tissues and also on pancreatic islet cells. As a negative co-stimulator of the immune system, it is an immune checkpoint protein. The interaction between these two proteins can inhibit the activity of T cells and promote the occurrence and development of diseases. Therefore, the expression levels and gene polymorphisms of PD-1 and PD-L1 may be closely related to the progression, prognosis of diabetes, and the effect of immunotherapy.

[0003] Currently, the detection of PD-1 and PD-L1 gene polymorphisms mainly relies on the following several techniques:

[0004] 1. Polymerase chain reaction (PCR) combined with sequencing: This is one of the most commonly used methods. Specific gene fragments are amplified by PCR, and then gene sequencing is performed to detect polymorphic sites. For example, the detection of SNPs (single nucleotide polymorphisms) in the PD-1 gene has been applied in many studies.

[0005] 2. Restriction fragment length polymorphism (RFLP): The size differences of gene fragments are analyzed through specific restriction enzyme cleavage sites to judge polymorphisms. This method is simple and low-cost, but it has limitations in detecting complex polymorphic sites.

[0006] 3. Real-time quantitative PCR (qPCR): It is used to identify the relative expression levels of specific genotypes and can provide accurate quantitative information, but usually requires a large number of samples and a high technical level.

[0007] 4. Single nucleotide polymorphism gene chip: This technology can detect multiple SNPs simultaneously, improving the detection throughput. However, it has a high cost and a complex experimental design.

[0008] Existing technologies mainly focus on detecting the expression of PD-1 and PD-L1, lacking efficient and accurate detection tools for their gene polymorphisms. This deficiency in technology has restricted the process of personalized medicine in diabetes treatment. Therefore, there is an urgent need to develop corresponding kits for detecting gene polymorphisms. Summary of the Invention

[0009] The object of the present invention is to provide a novel PD-1 and PD-L1 gene polymorphism detection kit, which can quickly and accurately detect specific polymorphisms of PD-1 and PD-L1 genes, so as to provide a basis for individualized treatment plans for diabetic patients in immunotherapy, improve the effect of immunotherapy, and improve the prognosis of patients.

[0010] A PD-1 and PD-L1 gene polymorphism detection kit of the present invention is designed to quickly and accurately detect the rs34819629 locus of the PD-1 gene and the rs2297137 locus of the PD-L1 gene. The genotypes of the PD-1 locus rs34819629 are CC, CT and TT, and the three genotypes of the PD-L1 locus rs2297137 are GG, AG and AA. This kit consists of the following main parts:

[0011] (1) Primer design, the specific synthetic DNA primer sequences for the rs34819629 locus of the PD-1 gene and the rs2297137 locus of the PD-L1 gene are as follows:

[0012] The upstream primer sequence for the rs34819629 locus is: 5'-CACCTGGGGAATGGTGAACG-3';

[0013] The downstream primer sequence for the rs34819629 locus is: 5'-TTCACCTGGGGAATGGTGAGCA-3';

[0014] The upstream primer sequence for the rs2297137 locus is: 5'-TGCAAAGGCATTCCACTGTTCAAGAG-3';

[0015] The downstream primer sequence for the rs2297137 locus is: 5'-TTTGCAAAGGCATTCCACTGTTCAAGAA-3';

[0016] The primer design is based on the gene sequences of the target loci, aiming to specifically amplify the fragments containing polymorphic loci to ensure the specificity and accuracy of detection;

[0017] (2) PCR amplification reagents:

[0018] The PCR reaction buffer is: 10xPCR Buffer containing Mg 2+ ;

[0019] dNTPs: including four deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP, which are used for DNA replication;

[0020] The DNA polymerase is: Taq DNA polymerase or high-fidelity DNA polymerase;

[0021] Template DNA: Genomic DNA extracted from a patient's blood sample, used as a template for amplification.

[0022] The method for using a kit for detecting PD-1 and PD-L1 gene polymorphisms according to the present invention comprises the following steps:

[0023] Step 1: Sample treatment:

[0024] 1.1 DNA extraction: Extract genomic DNA from the peripheral blood sample of a diabetic patient as a template for amplification;

[0025] 1.2 DNA quality detection: Detect the DNA concentration and purity to ensure that the DNA quality is sufficient for PCR reaction, and the DNA concentration is greater than 30 ng / μL.

[0026] Step 2: DNA lysis:

[0027] The reaction system is 10 μL, containing 4 μL of DNA sample, 2.5 μL of 4×DNA lysis buffer (Lysis Buffer), and supplemented to 10 μL with double-distilled water (ddH2O);

[0028] Operation steps: Mix the reaction system and centrifuge for 30 s at a speed of 3000 rpm; after centrifugation, transfer to a PCR instrument, denature at 98°C for 5 min, and immediately place on ice for 5 min for standby.

[0029] Step 3: Preparation of ligation reaction premix:

[0030] The premix is 10 μL / well, containing 2 μL of 10×ligase buffer (Ligase Buffer), 0.5 μL of ligase (Ligase), 1 μL of probe mixture (Probe Mix), and 6.5 μL of double-distilled water (ddH2O);

[0031] Operation steps: Mix and centrifuge the substances in the premix on ice, and dispense into pre-cooled PCR tubes.

[0032] Step 4: Ligation reaction:

[0033] The reaction system is 20 μL, containing 10 μL of lysed DNA and 10 μL of ligation premix;

[0034] The reaction conditions of the PCR program are:

[0035] Pre-denaturation at 94°C for 1 min;

[0036] Ligation at 58°C for 4 h;

[0037] Termination at 72°C for 2 min;

[0038] Finally, store at 4°C;

[0039] Subsequent processing:

[0040] Immediate use: PCR after ice bath for 5 min;

[0041] Long-term storage: Store at -20°C and thaw at 4°C before use.

[0042] Step 5: Multiplex fluorescence PCR, amplify in two tubes:

[0043] Primer information:

[0044] Upstream of PD-1: 5'-CACCTGGGGAATGGTGAACG-3';

[0045] Downstream of PD-1: 5'-TTCACCTGGGGAATGGTGAGCA-3';

[0046] Upstream of PD-L1: 5'-TGCAAAGGCATTCCACTGTTCAAGAG-3';

[0047] Downstream of PD-L1: 5'-TTTGCAAAGGCATTCCACTGTTCAAGAA-3';

[0048] Premix 19 μL / tube, containing 10 μL of 2×PCR Master Mix, 1 μL of Primer Mix containing PD-1 / PD-L1 primers, and 8 μL of ddH2O;

[0049] PCR system is 20 μL, containing 19 μL of premix and 1 μL of ligation product;

[0050] PCR program reaction conditions are:

[0051]

[0052] Step 6: Sequence with ABI 3730xl DNA analyzer:

[0053] Operation steps:

[0054] 6.1. Dilute the PCR product 10-fold with ddH2O;

[0055] 6.2. Take 1 μL of the diluted product, 0.5 μL of Liz600, and 8.5 μL of Hi-Di formamide and mix well;

[0056] 6.3. Denature the mixture at 95°C for 5 min and then ice bath for 2 min;

[0057] Upper machine: POP-7 polymer, running in Fragment Analysis mode.

[0058] Step 7: Analyze the data using GeneMapper 4.1 genotyping analysis software:

[0059] Parameter settings:

[0060] The detection panel file needs to match the PD-1 / PD-L1 locus;

[0061] Peak height threshold ≥ 50 RFU;

[0062] Quality control standards:

[0063] Negative control: No amplification peak;

[0064] Positive control: All loci are detected.

[0065] Advantages of the kit of the present invention:

[0066] 1. High precision: Through specially designed primers and high-throughput sequencing technology, the gene polymorphisms of PD-1 and PD-L1 can be accurately identified;

[0067] 2. Simple operation: The operation process of the detection kit is simple and can be carried out in a conventional laboratory environment, facilitating application and popularization;

[0068] 3. Reduce the detection cost: By optimizing the composition of the reagent and the detection process, the production and use costs of the kit are reduced, making it more economical, thus promoting its widespread application in clinics. Description of the drawings

[0069] Figure 1 It is the melting curve graph in Example 2;

[0070] Figure 1 A is the melting curve graph of three genotypes (TT, CC, CT) at the rs34819629 locus of the PD-1 gene;

[0071] Figure 1 B is the melting curve graph of three genotypes (GG, AA, AG) at the rs229737 locus of the PD-L1 gene. Detailed implementation manners

[0072] The following further illustrates the content of the present invention in conjunction with examples and drawings, but does not limit the present invention.

[0073] Example 1

[0074] A kit for detecting PD-1 and PD-L1 gene polymorphisms, the composition and key parameters of the kit include:

[0075] (1) The specific synthetic DNA primer sequences for the rs34819629 locus of the PD-1 gene and the rs2297137 locus of the PD-L1 gene are as follows:

[0076] For the rs34819629 locus of the PD-1 gene:

[0077] The upstream primer sequence is: 5'-CACCTGGGGAATGGTGAACG-3' (concentration 0.2 μM);

[0078] The downstream primer sequence is: 5'-TTCACCTGGGGAATGGTGAGCA-3' (concentration 0.2 μM);

[0079] For the rs2297137 locus of the PD-L1 gene:

[0080] The upstream primer sequence is: 5'-TGCAAAGGCATTCCACTGTTCAAGAG-3' (concentration 0.2 μM);

[0081] The downstream primer sequence is: 5'-TTTGCAAAGGCATTCCACTGTTCAAGAA-3' (concentration 0.2 μM);

[0082] (2) PCR amplification reagents:

[0083] 10×PCR Buffer (containing 15 mM MgCl2);

[0084] dNTPs mixture (each 2.5 mM);

[0085] Hot start Taq DNA polymerase (5 U / μL);

[0086] Specific probe Mix (containing FAM / HEX double labeling).

[0087] Example 2

[0088] A method for using a kit for detecting polymorphisms of PD-1 and PD-L1 genes, comprising the following steps

[0089] Step 1: Sample processing:

[0090] 1.1. DNA extraction: Take 200 μL of EDTA-anticoagulated peripheral blood and extract DNA using the magnetic bead method;

[0091] 1.2. DNA quality detection:

[0092] Quality requirements: A260 / A280 = 1.8 - 2.0, concentration ≥ 30 ng / μL (Qubit quantification);

[0093] Concentration estimation: Compare with a DNA Marker of known concentration (such as λ-Hind III) to ensure that the sample concentration is 30 - 50 ng / μL. If the concentration does not meet the requirement, re-extract the sample or adjust the subsequent loading volume.

[0094] Electrophoresis verification: Take 1 μL of the DNA sample and perform electrophoresis (5 - 10 min, 100 V) in a 1% agarose gel (containing nucleic acid dye). Quality requirement: The main band should be clear (>10 kb) and there should be no degradation (no smearing band).

[0095] Step 2: DNA lysis: Reaction system (10 μL):

[0096] Component Volume (μL) DNA Sample 4 μL (≥120 ng) 4×DNALysis Buffer 2.5 μL (containing 0.4 M NaOH) <![CDATA[ddH2O]]> Make up to 10 μL

[0097] Operation steps:

[0098] Mix the reaction components and centrifuge briefly (3000 rpm, 30 s).

[0099] Denature at 98 °C for 5 min (using a PCR instrument), and immediately ice-bath for 5 min.

[0100] Centrifuge briefly and place on ice for standby (to avoid renaturation).

[0101] Step 3: Preparation of ligation reaction premix:

[0102] Composition of the premix (10 μL / reaction):

[0103] Reagent Volume (μL) 10×Ligase Buffer 2 μL (containing ATP) Ligase 0.5 μL (5 U) Probe Mix (containing PD-1 / PD-L1 primers) 1 μL <![CDATA[ddH2O]]> 6.5 μL Total Volume 10 μL

[0104] Preparation steps:

[0105] Operate on ice to avoid enzyme inactivation.

[0106] Vortex and mix well, then centrifuge briefly and aliquot into ice-precooled PCR tubes (10 μL / well).

[0107] Reaction conditions: 94 °C for 1 min → 58 °C for 4 h → 72 °C for 2 min;

[0108] Step 4: Ligation reaction:

[0109] Reaction system 20 μL: Ice-precooled lysed DNA (10 μL) + ligation premix (10 μL); PCR program:

[0110] Step Step Time Number of Cycles Pre-denaturation 94℃ 1 min 1 Ligation 58℃ 4h 4 Termination 72℃ 2 min 1 Storage 4℃ ∞ -

[0111] Subsequent treatment:

[0112] Immediate use: After ice-bathing for 5 min, directly perform PCR.

[0113] Long-term storage: Store at -20°C. Thaw at 4°C for 10 min before use and keep on ice bath for standby.

[0114] Step 5: Multiplex fluorescence PCR (amplify in two tubes, a total of 96 loci):

[0115] Primer information:

[0116] Upstream of PD-1: 5'-CACCTGGGGAATGGTGAACG-3';

[0117] Downstream of PD-1: 5'-TTCACCTGGGGAATGGTGAGCA-3';

[0118] Upstream of PD-L1: 5'-TGCAAAGGCATTCCACTGTTCAAGAG-3';

[0119] Downstream of PD-L1: 5'-TTTGCAAAGGCATTCCACTGTTCAAGAA-3';

[0120] Preparation of premix:

[0121] Reagent Volume (μL) 2×PCR Master Mix 10 μL (containing HotStart Taq) Primer Mix (containing PD-1 / PD-L1 primers) 1 μL (final concentration 0.2 μM) <![CDATA[ddH2O]]> 8 μL Total Volume 19 μL

[0122] Reaction system (20 μL): Premix (19 μL) + Ligation product (1 μL);

[0123] PCR program reaction conditions are:

[0124]

[0125] Step 6: Sequence with ABI 3730xl DNA Analyzer:

[0126] 6.1 Dilution and denaturation:

[0127] 6.1.1. Dilute the PCR product 10-fold with ddH2O;

[0128] 6.1.2. Mixing: Take the diluted product (1 μL) + Liz600 (0.5 μL) + Hi-Di formamide (8.5 μL) and mix well;

[0129] 6.1.3. Denature the mixture at 95°C for 5 min and immediately place on ice bath for 2 min;

[0130] 6.2 Loading onto the machine: Use POP-7 polymer and run on ABI 3730XL sequencer (standard Fragment Analysis mode).

[0131] Step 7: Perform data analysis with GeneMapper 4.1 genotyping analysis software:

[0132] Key parameters:

[0133] Peak height flux threshold: 50RFU

[0134] Allele typing criteria:

[0135] rs34819629: T peak (150bp) / C peak (150bp)

[0136] rs2297137: G peak (120 bp) / A peak (120 bp)

[0137] Melting curve analysis and graph interpretation, such as Figure 1 As shown:

[0138] like Figure 1 As shown in A, it is the melting curve of three genotypes (TT, CC, CT) of the rs34819629 site of the PD-1 gene:

[0139] TT-type samples showed a single high Tm peak;

[0140] The CC type samples showed low Tm peaks;

[0141] The CT type sample showed double peaks, corresponding to two Tm values, CC and TT, indicating a heterozygous type.

[0142] like Figure 1 B shows the melting curves of three genotypes (GG, AA, AG) at the rs229737 site of the PD-L1 gene:

[0143] GG type has a single high Tm peak;

[0144] AA type is a low Tm peak;

[0145] The AG type showed two Tm peaks, which is a typical characteristic of the heterozygous type.

[0146] Quality Control Standards:

[0147] Must-control indicators:

[0148] Positive control: All target sites must be detected;

[0149] Negative control: RFU values ​​of all channels <30;

[0150] Internal reference gene (ACTB): Ct value ≤ 28;

[0151] Performance parameters:

[0152] Detection limit: 5 ng / μL DNA;

[0153] Repeatability: CV < 5% (n = 20);

[0154] Specificity: Can distinguish single-base differences with 100% accuracy;

[0155] Typical detection results:

[0156] Detection of 149 type 2 diabetes patients showed that:

[0157] Genotype distribution of rs34819629:

[0158] TT type: 25 cases (16.80%);

[0159] CT type: 74 cases (49.70%);

[0160] CC type: 50 cases (33.60%);

[0161] Genotype distribution of rs2297137:

[0162] GG type: 54 cases (36.20%);

[0163] AG type: 71 cases (47.70%);

[0164] AA type: 24 cases (16.10%);

[0165] Note: All experimental data were statistically analyzed using SPSS 26.0. Measurement data were expressed as x±s, and count data were expressed as rate (%).

[0166] It was confirmed by the examples that when using the above parameter combination:

[0167] 1. The genotyping accuracy rate reached 100% (verified by Sanger sequencing);

[0168] 2. The within-batch coefficient of variation was only 3.2%;

[0169] 3. It can stably detect trace mutations of ≥5%;

[0170] 4. The whole detection time was controlled within 8 hours.

[0171] The method of using the PD-1 and PD-L1 gene polymorphism detection kit of the present invention. The examples gave a specific method of use. In actual use, such as the dosage of the reaction system, the raw materials can be added according to multiples of the dosage in the examples.

[0172] The kit of the present invention is an efficient tool for detecting specific polymorphisms of PD-1 and PD-L1 genes, with the characteristics of high precision, simple operation, and support for personalized medicine. Through this detection kit, targeted immunotherapy suggestions can be provided for patients, significantly improving the effectiveness of immunotherapy, and providing important gene information for related research, promoting the development of diabetes immunotherapy.

Claims

1. A PD-1 and PD-L1 gene polymorphism detection kit, characterized in that: The kit is used to detect the rs34819629 site of the PD-1 gene and the rs2297137 site of the PD-L1 gene, and the kit consists of the following main parts: (1) Primer design: The specific artificial synthetic DNA primer sequences for rs34819629 and rs2297137 sites are as follows: The upstream primer sequence of rs34819629 was: 5′-CACCTGGGGAATGGTGAACG-3′; The downstream primer sequence of rs34819629 was: 5′-TTCACCTGGGGAATGGTGAGCA-3′; The upstream primer sequence of rs2297137 was: 5′-TGCAAAGGCATTCCACTGTTCAAGAG-3′; The downstream primer sequence of rs2297137 was: 5′-TTTGCAAGGCATTCCACTGTTCAAGAA-3′; (2) PCR amplification reagents: PCR reaction buffer: containing Mg 2+ 10xPCR Buffer; dNTPs: including four deoxynucleotide triphosphates dATP, dCTP, dGTP and dTTP, used for DNA replication; DNA polymerase: Taq DNA polymerase or high-fidelity DNA polymerase; Template DNA: Genomic DNA extracted from the patient's blood sample, used as a template for amplification.

2. The method for using the PD-1 and PD-L1 gene polymorphism detection kit according to claim 1, characterized in that: The method of use comprises the following steps: Step 1: Sample processing: 1.

1. Extract DNA: Extract genomic DNA from peripheral blood samples of diabetic patients as a template for amplification; 1.

2. DNA quality test: Perform DNA concentration and purity tests to ensure that the DNA quality is sufficient for PCR reaction and the DNA concentration is greater than 30 ng / μL; Step 2: DNA lysis: The reaction system is 10 μL, including 4 μL of DNA sample, 2.5 μL of 4× DNA lysis buffer, and 10 μL made up with double distilled water (ddH2O); Operation steps: Mix the reaction system and centrifuge for 30 seconds at 3000 rpm; transfer to PCR instrument after centrifugation, denature at 98℃ for 5 minutes, and immediately put on ice for 5 minutes for later use; Step 3: Preparation of ligation reaction premix: The premix solution is 10 μL / well, including 2 μL of 10× Ligase Buffer, 0.5 μL of Ligase, 1 μL of Probe Mix, and 6.5 μL of double distilled water (ddH2O). Operation steps: Mix all substances in the premix on ice, centrifuge, and dispense into precooled PCR tubes; Step 4: Ligation reaction: The reaction system is 20 μL, including 10 μL of lysed DNA and 10 μL of ligation premix; Step 5: Multiplex fluorescence PCR, amplification in two tubes: Primer Information: PD-1 upstream: 5′-CACCTGGGGAATGGTGAACG-3′; PD-1 downstream: 5′-TTCACCTGGGGAATGGTGAGCA-3′; PD-L1 upstream: 5′-TGCAAAGGCATTCCACTGTTCAAGAG-3′; PD-L1 downstream: 5′-TTTGCAAAGGCATTCCACTGTTCAAGAA-3′; The master mix was 19 μL / tube, including 10 μL of 2× PCR master mix, 1 μL of primer premix containing PD-1 / PD-L1 primers, and 8 μL of ddH2O; PCR system 20 μL, including 19 μL premix and 1 μL ligation product; Step 6: Sequencing using ABI 3730xl DNA Analyzer: 6.1 Dilution and denaturation: 6.1.

1. Dilute the PCR product 10 times with ddH2O; 6.1.

2. Mixing: Take 1 μL of diluted product + 0.5 μL of Liz600 + 8.5 μL of Hi-Di formamide and mix well; 6.1.

3. Denature the mixture at 95°C for 5 min and immediately place in an ice bath for 2 min. 6.2 On-line: Using POP-7 polymer, ABI 3730XL sequencer was run in standard Fragment Analysis mode; Step 7: Analyze the data using GeneMapper 4.1 genotyping analysis software: Parameter settings: The detection combination panel file needs to match the PD-1 / PD-L1 sites; Peak height threshold ≥50RFU; Quality control standards: Negative control: no amplification peak; Positive control: all sites detected.

3. The method for using the PD-1 and PD-L1 gene polymorphism detection kit according to claim 2, characterized in that: The PCR program reaction conditions in step 4 of the method of use are: Pre-denaturation at 94°C for 1 min; Connect at 58°C for 4 hours; Terminate at 72°C for 2 min; Finally, store at 4°C; Subsequent processing: Immediate use: PCR after 5 min of ice bath; Long-term storage: Freeze at -20℃ and thaw at 4℃ before use.

4. The method for using the PD-1 and PD-L1 gene polymorphism detection kit according to claim 2, characterized in that: The PCR program reaction conditions in step 5 of the method of use are:

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