Primer group for detecting individualized medication gene locus polymorphism of hypoglycemic drug, kit containing primer group and application

Abstract

The invention relates to a primer group for detecting individualized medication gene locus polymorphism of hypoglycemic drugs, a kit containing the primer group and application, and belongs to the technical field of gene polymorphism detection. The primer group for the individualized medication gene locus polymorphism of the hypoglycemic drugs comprises at least one of 18 groups of primer pairs aiming at 18 SNP (Single Nucleotide Polymorphism) loci respectively, wherein the nucleotide sequences of reverse and forward primers of the 18 groups of primer pairs are SEQ ID NO: 1-SEQ ID NO: 36; the invention also comprises a UEP primer for the single base extension reaction, and the nucleotide sequence of the primer is selected from at least one of SEQ ID NO: 37-SEQ ID NO: 54. Based on the primer, the invention provides a kit containing the primer and an application method. The technical problems that the detection flux is limited or the biological information analysis and interpretation amount is large, and the detection cost is high are solved, the detection result can be obtained only through the trace genome DNA, the consumed time is short, the detection efficiency is high, the cost is remarkably reduced, and accurate medication of the hypoglycemic drug can be achieved.

Description

Technical Field

[0001] This invention belongs to the field of gene polymorphism detection technology, and particularly relates to a primer set for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs, a kit containing the primer set, and its application. Background Technology

[0002] Diabetes mellitus is a group of chronic metabolic diseases characterized by hyperglycemia. While diabetic patients appear to have elevated blood sugar, they also experience damage to the nervous system, microvessels, and macrovessels. Therefore, consistent medication is necessary to control blood sugar levels. However, this also brings side effects, such as: Repaglinide's adverse reactions may include hypoglycemia, abdominal pain, nausea (rare), diarrhea, vomiting, constipation, visual abnormalities, and liver abnormalities; the insulin sensitizer pioglitazone commonly causes hypoglycemia, edema, and osteoporosis; gastrointestinal reactions are the main adverse reaction of metformin, with common symptoms including diarrhea, abdominal pain, anorexia, nausea, vomiting, bloating, abdominal discomfort, and indigestion. Some patients may experience a metallic taste in their mouth after using it.

[0003] Individual differences in responses to hypoglycemic drugs are common, and their mechanisms are related to various factors such as genetics, sex, age, height, weight, lifestyle and dietary habits, concomitant medications, and disease state, with genetic factors being the most important. Currently, individualized medication for diabetic patients to avoid poor efficacy or severe side effects such as hypoglycemia has become a research hotspot in treatment. Studies in pharmacogenetics have shown that polymorphisms in drug metabolism-related gene loci are important factors causing individual differences in efficacy and toxicity. These gene loci are involved in a series of functions, including encoding drug-metabolizing enzymes, transporters, and targets. Therefore, detecting and analyzing these gene loci can help reduce adverse reactions, increase medication safety, improve drug efficacy, effectively control blood sugar, reduce complications, and shorten the drug trial cycle, avoiding the waste of time and money from repeated drug trials. These are particularly important for patients with hyperglycemia.

[0004] Furthermore, with the continuous development of molecular detection technologies, personalized medicine testing projects that detect multiple sites / multiple drugs in a single batch have become the mainstream trend. Traditional gene mutation detection methods, such as Sanger sequencing, pyrosequencing, and real-time fluorescence PCR, are no longer suitable due to their limited throughput; each batch can only detect mutations in a single gene or only a portion of the exons of a single gene. Next-generation sequencing (NGS) can obtain information on hundreds of pharmacogenomics-related genes in a single test, but it suffers from extremely large bioinformatics analysis and interpretation volumes and high testing costs. Therefore, based on the above shortcomings, providing a high-throughput, low-cost, and highly accurate personalized medicine gene locus polymorphism detection solution has become a pressing technical problem that needs to be solved. Summary of the Invention

[0005] (a) Technical problems to be solved In view of the above-mentioned shortcomings and deficiencies of the prior art, the present invention provides a primer set for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs, a kit containing the primer set, and its application. It solves the technical problems of limited detection throughput or large amount of bioinformatics analysis and interpretation, and high detection cost. The present invention provides 18 gene loci related to hypoglycemic drugs, involving 17 drugs, with high sensitivity, requiring only a trace amount of genomic DNA to obtain detection results, short detection time, high detection efficiency, and significantly reduced cost, enabling precise medication of hypoglycemic drugs.

[0006] (II) Technical Solution This invention proposes a primer set for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs, a kit containing the primer set, and its applications.

[0007] One objective of this invention is to provide a primer set for detecting gene locus polymorphisms related to personalized medication of hypoglycemic drugs, including an amplification primer set for multiplex PCR amplification of hypoglycemic drug-related genes; the amplification primer set includes forward and reverse primer pairs targeting the following SNP loci: rs2237895, rs628031, rs2289669, rs7754840, rs889299, rs5219, rs10811661, rs7756992, rs2241766, rs1057910, rs4149056, rs17584499, rs6923761, rs10305420, rs12943590, rs1799853, rs296766, and rs10494366; the SNP loci proposed in this invention are used to amplify gene loci related to 18 hypoglycemic drugs.

[0008] in, The primer pair targeting the rs2237895 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:1 and a forward primer of the nucleotide sequence shown in SEQ ID NO:2. The primer pair targeting the rs628031 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:3 and a forward primer of the nucleotide sequence shown in SEQ ID NO:4. The primer pair targeting the rs2289669 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:5 and a forward primer of the nucleotide sequence shown in SEQ ID NO:6. The primer pair targeting the rs7754840 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:7 and a forward primer of the nucleotide sequence shown in SEQ ID NO:8. The primer pair targeting the rs889299 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:9 and a forward primer of the nucleotide sequence shown in SEQ ID NO:10. The primer pair targeting the rs5219 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:11 and a forward primer for the nucleotide sequence shown in SEQ ID NO:12. The primer pair targeting the rs10811661 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:13 and a forward primer for the nucleotide sequence shown in SEQ ID NO:14. The primer pair targeting the rs7756992 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:15 and a forward primer of the nucleotide sequence shown in SEQ ID NO:16. The primer pair targeting the rs2241766 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:17 and a forward primer of the nucleotide sequence shown in SEQ ID NO:18. The primer pair targeting the rs1057910 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:19 and a forward primer for the nucleotide sequence shown in SEQ ID NO:20. The primer pair targeting the rs4149056 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:21 and a forward primer for the nucleotide sequence shown in SEQ ID NO:22. The primer pair targeting the rs17584499 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:23 and a forward primer for the nucleotide sequence shown in SEQ ID NO:24. The primer pair targeting the rs6923761 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:25 and a forward primer for the nucleotide sequence shown in SEQ ID NO:26. The primer pair targeting the rs10305420 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:27 and a forward primer for the nucleotide sequence shown in SEQ ID NO:28. The primer pair targeting the rs12943590 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:29 and a forward primer for the nucleotide sequence shown in SEQ ID NO:30. The primer pair targeting the rs1799853 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO:31 and a forward primer of the nucleotide sequence shown in SEQ ID NO:32. The primer pair targeting the rs296766 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:33 and a forward primer for the nucleotide sequence shown in SEQ ID NO:34. The primer pair for the rs10494366 site consists of a reverse primer for the nucleotide sequence shown in SEQ ID NO:35 and a forward primer for the nucleotide sequence shown in SEQ ID NO:36.

[0009] Furthermore, it also includes UEP primers for single-base extension reactions; The UEP primers include primers targeting the following SNP sites: rs2237895, rs628031, rs2289669, rs7754840, rs889299, rs5219, rs10811661, rs7756992, rs2241766, rs1057910, rs4149056, rs17584499, rs6923761, rs10305420, rs12943590, rs1799853, rs296766, and rs10494366; in, The nucleotide sequence of the UEP primer for rs2237895 is shown in SEQ ID NO:37; The nucleotide sequence of the UEP primer for rs628031 is shown in SEQ ID NO:38; The nucleotide sequence of the UEP primer for rs2289669 is shown in SEQ ID NO:39; The nucleotide sequence of the UEP primer targeting rs7754840 is shown in SEQ ID NO:40; The nucleotide sequence of the UEP primer for rs889299 is shown in SEQ ID NO:41; The nucleotide sequence of the UEP primer for rs5219 is shown in SEQ ID NO: 42; The nucleotide sequence of the UEP primer for rs10811661 is shown in SEQ ID NO:43; The nucleotide sequence of the UEP primer for rs7756992 is shown in SEQ ID NO:44; The nucleotide sequence of the UEP primer for rs2241766 is shown in SEQ ID NO:45; The nucleotide sequence of the UEP primer for rs1057910 is shown in SEQ ID NO:46; The nucleotide sequence of the UEP primer for rs4149056 is shown in SEQ ID NO:47; The nucleotide sequence of the UEP primer for rs17584499 is shown in SEQ ID NO:48; The nucleotide sequence of the UEP primer for rs6923761 is shown in SEQ ID NO:49; The nucleotide sequence of the UEP primer for rs10305420 is shown in SEQ ID NO:50; The nucleotide sequence of the UEP primer targeting rs12943590 is shown in SEQ ID NO:51; The nucleotide sequence of the UEP primer for rs1799853 is shown in SEQ ID NO:52; The nucleotide sequence of the UEP primer for rs296766 is shown in SEQ ID NO:53; The nucleotide sequence of the UEP primer for rs10494366 is shown in SEQ ID NO:54.

[0010] The second objective of this invention is to provide a kit for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs.

[0011] Furthermore, the kit includes a set of primers for multiplex PCR amplification of hypoglycemic drug-related genes and UEP primers for single-base extension reactions.

[0012] Furthermore, the kit includes a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase purification reaction system, and a single-base extension reaction system; the multiplex PCR amplification reaction system includes multiplex PCR amplification primers, PCR buffer, deoxyribonucleoside triphosphate mixture, DNA polymerase, and MgCl2; the shrimp alkaline phosphatase purification reaction system includes SAP enzyme and SAP buffer; the single-base extension reaction system includes UEP primers, iPLEX Buffer Plus, iPLEX Terminationmix, and iPLEX Pro Enzyme.

[0013] The third objective of this invention is to provide a primer set or a kit for detecting gene locus polymorphisms in personalized medicine for hypoglycemic agents, and its application in detecting gene locus polymorphisms in personalized medicine for hypoglycemic agents. The fourth objective of this invention is to provide a method for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs using a kit for detecting such polymorphisms, comprising the following steps: S1. Extract template DNA from the sample, prepare a multiplex PCR amplification reaction system, perform multiplex PCR amplification reaction, and obtain multiplex PCR amplification products; S2. The multiplex PCR amplification product is purified by SAP enzyme to obtain the purified product; S3. The purified product is subjected to a single-base extension reaction to obtain the extended product; S4. Detect the extended product to obtain the gene site polymorphism of personalized medication for hypoglycemic drugs.

[0014] Further, in S1, the multiplex PCR amplification reaction system includes ultrapure water, PCR buffer, MgCl2, dNTP, P mix, PCR enzyme, and DNA template; the P mix is ​​a mixture of primer sequences shown in SEQ ID NO:1 to SEQ ID NO:36.

[0015] Furthermore, the SAP purification reaction system includes ultrapure water, SAP Buffer, SAP Enzyme, and amplification products.

[0016] Furthermore, the single-base extension reaction system includes ultrapure water, iPLEX Buffer Plus, iPLEXTermination mix, UEP Mix, iPLEX Pro Enzyme, and purified product; the UEP Mix is ​​a mixture of primer sequences shown in SEQ ID NO:37 to SEQ ID NO:54.

[0017] Furthermore, the hypoglycemic drugs include liraglutide, alogliptin, dipeptidyl peptidase-4 (DPP-4) inhibitors, linagliptin, saxagliptin, vildagliptin, sitagliptin, glibenclamide, glipizide, glimepiride, gliclazide, nateglinide, repaglinide, troglitazone, pioglitazone, rosiglitazone, and metformin.

[0018] The reference genome version used in this invention is GRCh38 / hg38.

[0019] (III) Beneficial Effects The hypoglycemic drug personalized drug gene locus polymorphism detection kit of the present invention has the advantages of high accuracy, high throughput, low cost and short cycle. Compared with conventional Sanger sequencing, pyrosequencing and real-time fluorescence PCR technology, it has a significant high throughput advantage.

[0020] Furthermore, compared to next-generation sequencing (NGS), this invention completes the testing of a batch of samples in just 8-10 hours, significantly reducing testing time, efficiency, and cost. The kit of this invention can detect 18 gene loci related to hypoglycemic drugs, involving 17 drugs. It boasts high sensitivity and requires only a trace amount of genomic DNA for pooled testing within the same system. The test results can guide clinicians in selecting appropriate drugs and dosages, enabling patients to achieve optimal treatment outcomes while minimizing adverse drug reactions, truly achieving the goal of precision medicine. Attached Figure Description

[0021] Figure 1 This is the mass spectrum of sample 90 at site rs10811661 in this invention; Figure 2 This is the Sanger sequencing map of the rs10811661 site of sample 90 in this invention; Figure 3 This is the mass spectrum of sample 90 at the rs5219 site in this invention; Figure 4 This is a Sanger sequencing image of the rs5219 site in sample 90 of this invention; Figure 5 This is the mass spectrum of sample 90, rs6923761. Figure 6 This is the Sanger sequencing map of the rs6923761 site in sample 90 of this invention; Figure 7 This is the mass spectrum of sample 90 at site rs628031 in this invention; Figure 8 This is a Sanger sequencing image of the rs628031 site of sample 90 in this invention; Figure 9 This is the mass spectrum of sample 88 from the present invention at locus rs10305420; Figure 10 This is the Sanger sequencing map of the rs10305420 site of sample 88 of this invention; Figure 11 This is the mass spectrum of sample 88, rs12943590. Figure 12 This is the Sanger sequencing map of the rs12943590 site of sample 88 of this invention; Figure 13 This is the mass spectrum of sample 88 at site rs4149056 of the present invention; Figure 14 This is the Sanger sequencing map of the rs4149056 site of sample 88 of this invention; Figure 15 This is the mass spectrum of sample 88 (rs889299) of the present invention. Figure 16 This is a Sanger sequencing image of the rs889299 site of sample 88 of this invention. Detailed Implementation

[0022] To better explain and facilitate understanding of the present invention, the present invention will be described in detail below with reference to the accompanying drawings and specific embodiments.

[0023] This invention proposes a primer set for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs, a kit containing the primer set, and its application. The mechanism is as follows: This invention proposes a method for detecting gene locus polymorphisms in personalized hypoglycemic drugs. Based on extensive pharmacogenomics research, this method identifies gene loci associated with responses to specific hypoglycemic drugs. Genomic DNA is extracted from the sample during processing. Detection is performed using a primer set, which consists of a series of carefully designed short-chain DNA sequences specifically designed to identify and amplify DNA fragments containing the target hypoglycemic loci. Finally, specific genotype results are obtained using a detection instrument to guide clinical medication. This method integrates multiple steps, including complex sample DNA extraction, PCR amplification, and genotyping, into a single closed kit, achieving standardization and automation. Through optimized multiplex PCR primer design, multiple loci of different hypoglycemic-related genes are simultaneously amplified and detected in a single reaction system, connecting genetic data with clinical practice and enabling the implementation of personalized medication.

[0024] Based on the above mechanism, this invention proposes a method for detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs, comprising the following steps: (1) Multiplex PCR amplification reaction: Template DNA was extracted from the sample, and a multiplex PCR amplification reaction system was prepared. Multiplex PCR amplification was performed to obtain the multiplex PCR amplification product. The multiplex PCR amplification reaction system was 5 μL and included the following components: 0.8 μL of ultrapure water, 0.5 μL of 10×PCR Buffer, 0.4 μL of 25 mM MgCl2, 0.1 μL of dNTP, 1 μL of P Mix, 0.2 μL of PCR Enzyme, and 2 μL of DNA template; The P mix is ​​a mixture of primer sequences from SEQ ID NO:1 to SEQ ID NO:36, with each primer having a concentration of 0.5 μM in the multiplex PCR amplification reaction system; (2) Purification reaction of shrimp alkaline phosphatase: The multiplex PCR amplification products were purified using SAP enzyme. The SAP purification reaction system consisted of the following components: 1.53 μL of ultrapure water, 0.17 μL of SAP Buffer, 0.3 μL of SAP Enzyme, and 5 μL of amplification product, for a total of 7 μL of reaction system. The purified product was obtained after the reaction. (3) Single base extension reaction The purified product from step (2) was subjected to a single-base extension reaction. The single-base extension reaction system included the following components: 0.62 μL of ultrapure water, 0.2 μL of iPLEX Buffer Plus, 0.2 μL of iPLEX Termination mix, 0.94 μL of UEP Mix, 0.04 μL of iPLEX Pro Enzyme, and 7 μL of purified product, for a total of 9 μL of reaction system; the extension product was obtained after the reaction. UEP Mix is ​​a mixture of primer sequences of SEQ ID NO:37 to SEQ ID NO:54, with each primer having a concentration of 0.7 μM in the single-base extension reaction system; (4) Nucleic acid mass spectrometry detection and analysis 35 μL of deionized water was added to the extended product, and then the sample was analyzed using a nucleic acid mass spectrometer to obtain the gene locus polymorphism for personalized medication of hypoglycemic drugs. The nucleic acid mass spectrometer is typically a MALDI-TOF MS matrix-assisted laser desorption / ionization-time-of-flight mass spectrometer. The nucleic acid mass spectrometer detector converts the electrical signal into a visual peak pattern. Each genotype has a unique molecular weight, and the software automatically analyzes the corresponding base types to obtain the final genotyping result.

[0025] Preferably, the reaction conditions for the multiplex PCR amplification reaction are: 95°C for 2 minutes; 95°C for 30 seconds, 56°C for 30 seconds, 72°C for 60 seconds, for 45 cycles; then 72°C for 5 minutes.

[0026] Preferably, the purification reaction conditions are 37°C for 40 minutes and 80°C for 5 minutes.

[0027] Preferably, the reaction program for the single-base extension reaction is 94℃ for 30s, {94℃ for 5s, (41℃ for 5s, 80℃ for 5s) 5 internal cycles} 39 external cycles, and 72℃ for 3min.

[0028] Preferably, the hypoglycemic drugs of the present invention include 17 types: liraglutide, linagliptin, metformin, glibenclamide, repaglinide, pioglitazone, etc.

[0029] Preferably, the hypoglycemic drugs of the present invention include 15 genes: GLP1R, CDKAL1, SLCO1B1, NOS1AP, KCNJ11, SLC22A1, etc.; Preferably, the gene loci of the hypoglycemic drugs of the present invention include rs10305420, rs6923761, rs7754840, rs10494366, rs5219, rs889299, rs2237895, rs628031, rs2289669, rs10811661, rs7756992, rs2241766, rs1057910, rs4149056, rs17584499, rs12943590, rs1799853, and rs296766, etc.

[0030] In summary, this invention first performs PCR amplification using self-designed primers, followed by SAP purification, and finally uses self-designed probes for target base extension. The obtained extension products are then analyzed by nucleic acid mass spectrometry to obtain relevant genotype information. This invention can test 96 samples (96-well plate) or 384 samples (384-well plate) in a batch, with each sample capable of detecting 18 gene loci. The hypoglycemic drug personalized medication gene locus polymorphism detection kit of this invention has the advantages of high accuracy, high throughput, low cost, and short cycle time. Compared with conventional Sanger sequencing, pyrosequencing, and real-time fluorescence PCR technology, it has a significant high-throughput advantage. Compared with next-generation sequencing (NGS), this invention can complete a batch of samples in only 8-10 hours, which is shorter and improves detection efficiency, while also being more affordable. The kit proposed in this invention can detect 18 gene loci related to hypoglycemic drugs, involving 17 drugs. It has high sensitivity and only requires a trace amount of genomic DNA to obtain detection results. It can be used to guide clinical selection of appropriate drugs. By using appropriate drug dosages, patients can obtain the best treatment effect while reducing adverse drug reactions, thus achieving the goal of precision medicine.

[0031] Example 1 An experiment for detecting SNP sites in DNA samples to be tested. One whole blood sample was collected from each of three volunteers, and the 18 SNP loci in these three samples were detected. Genomic DNA was first extracted from the whole blood of the three volunteers, followed by multiplex PCR, SAP purification, and single-base extension reaction. The products were then detected by nucleic acid mass spectrometry. Finally, the data were analyzed using relevant software to determine the genotypes of the 18 SNP loci in each sample.

[0032] result : Meanwhile, the mass spectrometry results were validated using a first-generation sequencing method. The validation results showed that the results of the method of this invention and the first-generation sequencing method had a 100% agreement rate.

[0033] Example 2 Provide a multiplex PCR amplification reaction experiment The PCR amplification system for a single sample consisted of: 0.8 μL ultrapure water, 0.5 μL 10x PCR Buffer (PCR Reagent And Spectro CHIP Kit, Agena Bioscience, catalog number 21327M), 0.4 μL 25 mM MgCl2, 0.1 μL dNTPs, 1 μL P Mix, 0.2 μL PCR Enzyme (PCR Reagent And Spectro CHIP Kit, Agena Bioscience, catalog number 21327M), and 2 μL DNA template, for a total reaction volume of 5 μL. The reaction conditions were: 95℃ for 2 minutes; 95℃ for 30 seconds, 56℃ for 30 seconds, 72℃ for 60 seconds, for 45 cycles; followed by 72℃ for 5 minutes. The primers for P Mix were SEQ ID NO. 1 to SEQ ID NO. 36 listed in Table 1, with each primer at a concentration of 0.5 μM. The DNA template was extracted using the Kangwei Century CWE960 BloodDNA Kit.

[0034] result : The multiplex PCR primer sequences are shown in Table 1.

[0035] Table 1: Primer sequence listing for multiplex PCR

[0036] In this invention, the correspondence between individualized hypoglycemic drug administration and gene loci is shown in Table 2.

[0037] Table 2: List of Correspondences between Personalized Hypoglycemic Drug Use and Gene Loci

[0038] Example 3 This provides the purification reaction, single-base extension reaction experiment, and nucleic acid mass spectrometry detection and analysis results of shrimp alkaline phosphatase (SAP). (1) Purification reaction of shrimp alkaline phosphatase (SAP) Add 2 μL of SAP mixture to each of the above amplification reaction wells, including: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, and 0.3 μL SAP Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), to obtain the SAP purification system: 1.53 μL ultrapure water, 0.17 μL SAP Buffer, 0.3 μL SAP Enzyme, and 5 μL of the first step PCR amplification product, for a total reaction volume of 7 μL. The purification reaction conditions were 37℃ for 40 minutes and 80℃ for 5 minutes.

[0039] (2) Monobase extension reaction After the purification reaction, 2 μL of iPLEX reaction system was added to each well to obtain the extension reaction system as follows: 0.62 μL ultrapure water, 0.2 μL iPLEX Buffer Plus (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), 0.2 μL iPLEX Termination mix (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), 0.94 μL UEP Mix, 0.04 μL iPLEX Pro Enzyme (iPLEX Pro kit, purchased from Agena Bioscience, catalog number: 10212), and 7 μL of the second-step purified product, for a total of 9 μL of reaction system. The extension UEP Mix sequence is SEQ ID NO.37 to SEQ ID NO.54 in Table 3, and the concentration of each primer is 0.7 μM. The reaction conditions are shown in Table 4.

[0040] Table 3: Extended UEP Mix Sequence List

[0041] Table 4: List of Reaction Conditions

[0042] (3) Nucleic acid mass spectrometry detection and analysis Add 35 μL of deionized water to the extension product. Place the sample in a MALDI-TOF MS matrix-assisted laser desorption / ionization time-of-flight mass spectrometer. Detection parameters are set as follows: Wells to Process - Automatic, Start Disoense Condition - 1000, Resin Volume - 13, Shot - 30, Maximum Acquisitions - 9, Minimum Good Spectra and Maximum Good Spectra - 5. Also check Tum Off Hy After Analysis, Transfer Resin to MTP, Dispense Samples onto Chips, Transfer chips to Analyzer, and Analyze Chips. The mass spectrometer spotter then spots the extension product onto the SpectroCHIP chip for detection. After detection, the detector converts the electrical signal into a visual peak pattern. Each genotype has a unique molecular weight, and the software automatically analyzes the corresponding base types to obtain the final genotyping result.

[0043] result : The results of the three test samples are shown in Tables 5-7. The test results show that the kit proposed in this invention can accurately detect the 18 SNP genotypes.

[0044] Table 5: Genotyping list of sample No. 88

[0045] Table 6: Genotyping list of sample No. 89

[0046] Table 7: Genotyping list of sample No. 90

[0047] Sanger sequencing was performed on identical samples 88-90, and the sequencing and mass spectrometry results were completely identical. Due to the large number of loci involved, only a comparison of representative locus detection patterns from samples 90 and 88 is shown. Figures 1-16 As shown.

[0048] Figure 1 This is the mass spectrometry result for the rs10811661 site of sample 90; the result is shown in the box. Figure 2 This is the Sanger sequencing result of the rs10811661 locus in sample 90; the result is shown in the box. Figure 3 This is the mass spectrometry result for the rs5219 site of sample 90; the result is shown in the box. Figure 4 This is the Sanger sequencing result of the rs5219 locus in sample 90; the result is shown in the box. Figure 5 This is the mass spectrometry result for the rs6923761 site of sample 90; the result is shown in the box. Figure 6 This is the Sanger sequencing result of the rs6923761 locus in sample 90, and the result is shown in the box. Figure 7 This is the mass spectrometry result of the rs628031 site in sample 90; the result is shown in the box. Figure 8 This is the Sanger sequencing result of the rs628031 locus in sample 90; the result is shown in the box. Figure 9 This is the mass spectrometry result for sample 88 at locus rs10305420; the result is shown in the box. Figure 10 This is the Sanger sequencing result of sample 88 at locus rs10305420; the result is shown in the box. Figure 11 This is the mass spectrometry result for the rs12943590 locus of sample 88; the result is shown in the box. Figure 12 This is the Sanger sequencing result of the rs12943590 locus in sample 88; the result is shown in the box. Figure 13This is the mass spectrometry result for the rs4149056 site of sample 88; the result is shown in the box. Figure 14 This is the Sanger sequencing result of the rs4149056 locus in sample 88; the result is shown in the box. Figure 15 This is the mass spectrometry result of sample 88 at locus rs889299; the result is shown in the box. Figure 16 This is the Sanger sequencing result of the rs889299 locus in sample 88, and the result is shown in the box.

[0049] Figures 1-16 The results showed that the detection method of the present invention was completely consistent with the results of Sanger sequencing, which indicates that the primer set and kit provided by the present invention have high accuracy, sensitivity and reliability in detecting gene locus polymorphisms in personalized medication of hypoglycemic drugs.

[0050] Finally, it should be noted that the above embodiments are only used to illustrate the technical solutions of the present invention, and not to limit them. Although the present invention has been described in detail with reference to the foregoing embodiments, those skilled in the art should understand that modifications can still be made to the technical solutions described in the foregoing embodiments, or equivalent substitutions can be made to some or all of the technical features. These modifications or substitutions, or combinations of technical features in the above embodiments that do not conflict with each other, can be made in accordance with the manner described in the embodiments. These modifications, substitutions or combinations do not cause the essence of the corresponding technical solutions to deviate from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer set for detecting polymorphism of a gene site for individualized medication of hypoglycemic drugs, characterized by, The amplification primer set for performing multiplex PCR amplification of hypoglycemic drug related genes; The amplification primer set comprises forward and reverse primer pairs for the following SNP sites: rs2237895, rs628031, rs2289669, rs7754840, rs889299, rs5219, rs10811661, rs7756992, rs2241766, rs1057910, rs4149056, rs17584499, rs6923761, rs10305420, rs12943590, rs1799853, rs296766 and rs10494366, respectively; wherein, The primer pair for the rs2237895 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 1 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 2; The primer pair for the rs628031 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 3 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 4; The primer pair for the rs2289669 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 5 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 6; The primer pair for the rs7754840 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 7 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 8; The primer pair for the rs889299 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 9 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 10; The primer pair for the rs5219 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 11 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 12; The primer pair for the rs10811661 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 13 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 14; The primer pair for the rs7756992 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 15 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 16; The primer pair for the rs2241766 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 17 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 18; The primer pair for the rs1057910 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 19 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 20; The primer pair for the rs4149056 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 21 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 22; The primer pair for the rs17584499 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 23 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 24; The primer pair for the rs6923761 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 25 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 26; The primer pair for the rs10305420 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 27 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 28; The primer pair for the rs12943590 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 29 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 30; The primer pair for the rs1799853 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 31 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 32; The primer pair for the rs296766 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 33 and a forward primer of the nucleotide sequence shown in SEQ ID NO: 34; The primer pair for the rs10494366 site consists of a reverse primer of the nucleotide sequence shown in SEQ ID NO: 35 and a forward primer of the nucleotide sequence shown in SEQ ID NO:

36.

2. The primer set for detecting hypoglycemic drug individualized medication genetic site polymorphism according to claim 1, characterized in that, The UEP primers for the single base extension reaction are also included; The UEP primers include primers for the following SNP sites: rs2237895, rs628031, rs2289669, rs7754840, rs889299, rs5219, rs10811661, rs7756992, rs2241766, rs1057910, rs4149056, rs17584499, rs6923761, rs10305420, rs12943590, rs1799853, rs296766 and rs10494366, respectively; The nucleotide sequence of the UEP primer for rs2237895 is shown in SEQ ID NO: 37; The nucleotide sequence of the UEP primer for rs628031 is shown in SEQ ID NO: 38; The nucleotide sequence of the UEP primer for rs2289669 is shown in SEQ ID NO: 39; The nucleotide sequence of the UEP primer for rs7754840 is shown in SEQ ID NO: 40; The nucleotide sequence of the UEP primer for rs889299 is shown in SEQ ID NO: 41; The nucleotide sequence of the UEP primer for rs5219 is shown in SEQ ID NO: 42; The nucleotide sequence of the UEP primer for rs10811661 is shown in SEQ ID NO: 43; ​ The nucleotide sequence of the UEP primer for rs7756992 is shown as SEQ ID NO: 44; The nucleotide sequence of the UEP primer for rs2241766 is shown as SEQ ID NO: 45; The nucleotide sequence of the UEP primer for rs1057910 is shown as SEQ ID NO: 46; The nucleotide sequence of the UEP primer for rs4149056 is shown as SEQ ID NO: 47; The nucleotide sequence of the UEP primer for rs17584499 is shown as SEQ ID NO: 48; The nucleotide sequence of the UEP primer for rs6923761 is shown as SEQ ID NO: 49; The nucleotide sequence of the UEP primer for rs10305420 is shown as SEQ ID NO: 50; The nucleotide sequence of the UEP primer for rs12943590 is shown as SEQ ID NO: 51; The nucleotide sequence of the UEP primer for rs1799853 is shown as SEQ ID NO: 52; The nucleotide sequence of the UEP primer for rs296766 is shown as SEQ ID NO: 53; The nucleotide sequence of the UEP primer for rs10494366 is shown as SEQ ID NO:

54.

3. A kit for detecting a polymorphism of a gene site for individualizing hypoglycemic drug medication, characterized by, The primer set for detecting the polymorphism of the genetic locus for individualized medication of hypoglycemic drugs according to claim 1 or 2.

4. The kit for detecting hypoglycemic drug individualized gene site polymorphism according to claim 3, characterized in that, The kit comprises a multiplex PCR amplification reaction system, a shrimp alkaline phosphatase purification reaction system, and a single base extension reaction system; The multiplex PCR amplification reaction system comprises multiplex PCR amplification primers, a PCR buffer, a deoxyribonucleotide triphosphate mixture, a DNA polymerase, and MgCl2; The shrimp alkaline phosphatase purification reaction system comprises a SAP enzyme and a SAP buffer; The single base extension reaction system comprises UEP primers, iPLEX Buffer Plus, iPLEX Termination mix, and iPLEX Pro Enzyme.

5. Use of the primer set for detecting the polymorphism of the genetic locus for individualized medication of hypoglycemic drugs according to claim 1 or 2 or the kit for detecting the polymorphism of the genetic locus for individualized medication of hypoglycemic drugs according to any one of claims 3-5 in detecting the polymorphism of the genetic locus for individualized medication of hypoglycemic drugs.

6. A method for detecting polymorphism of a gene site for individualized medication of hypoglycemic drugs using the kit for detecting polymorphism of a gene site for individualized medication of hypoglycemic drugs according to any one of claims 3 to 5, characterized by, The method comprises the following steps: S1, extracting template DNA from an isolated sample, preparing a multiplex PCR amplification reaction system, performing a multiplex PCR amplification reaction, and obtaining a multiplex PCR amplification product; S2, purifying the multiplex PCR amplification product by a SAP enzyme to obtain a purified product; S3, performing a single base extension reaction on the purified product to obtain an extension product; S4, detecting the extension product to obtain the polymorphism of the genetic locus for individualized medication of hypoglycemic drugs.

7. Use according to claim 6, characterized in that, In S1, the multiplex PCR amplification reaction system comprises ultrapure water, a PCR buffer, MgCl2, dNTP, P Mix, a PCR enzyme, and a DNA template; The P mix is a mixture of primer sequences shown in SEQ ID NO: 1-36.

8. Use according to claim 6, characterized in that, In S2, the SAP purification reaction system comprises ultrapure water, SAP Buffer, SAP Enzyme and amplification product.

9. Use according to claim 6, characterized in that, The single base extension reaction system comprises ultrapure water, iPLEX Buffer Plus, iPLEX Termination mix, UEP Mix, iPLEX Pro Enzyme and purified product. The UEP Mix is a mixture of primer sequences shown in SEQ ID NO: 37-54.

10. Use according to claim 6, characterized in that, The hypoglycemic drugs include liraglutide, linagliptin, metformin, glibenclamide, repaglinide and pioglitazone.

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