Primer group for detecting individualized medication gene locus, kit and application

Abstract

The invention discloses a primer probe set for noninvasive detection of individualized medication gene loci, a kit and application, and belongs to the field of biotechnology and molecular diagnosis. The sequences of the primer probe group are as shown in SEQ ID NO. 1 to SEQ ID NO. 51. The primer probe group is prepared into the kit, the kit comprises 7 tubes of independent premixed PCR reaction liquid, and high-throughput typing of 17 SNP loci of 12 drug genes is realized through a single-channel or multi-channel fluorescent PCR and melting curve analysis technology and a same PCR reaction program. An oral swab or a saliva sample is adopted and can be directly used as a template for gene detection after being treated by a nucleic acid lysis solution. Besides, the kit has the advantages of simplicity and convenience in operation, high sensitivity, low cost and the like, is suitable for detecting drug metabolism gene related types on an assembly line platform, and can be used for clinical early-stage rapid screening and accurate intervention.

Description

Technical Field

[0001] This invention belongs to the field of biotechnology and molecular diagnostics, specifically relating to a kit and detection method for the joint detection of multiple single nucleotide polymorphisms (SNPs) in an individual's genome that are associated with specific drug metabolism, transport, or targets using a multiplex PCR-melting curve method. It features simple operation, high specificity, and high sensitivity, and can be used to guide medication based on genotype results. Background Technology

[0002] Personalized medicine (precision medicine), as a core aspect of modern medicine, aims to achieve "the right drug and dosage for the right patient at the right time" by integrating key information such as a patient's genetic background and biomarkers. Extensive evidence suggests that up to 95% of the population carries at least one gene variant that can affect drug response. Polymorphisms in genes related to drug-metabolizing enzymes, transporters, and target molecules are the core genetic basis for individual differences in drug efficacy and toxicity. For example, the efficacy of the antiplatelet drug clopidogrel is highly dependent on the CYP2C19 genotype; the disease risk associated with statins is linked to genotypes such as ApoE and SLCO1B1; the emergency drug nitroglycerin may be ineffective in patients carrying the ALDH2 deficiency genotype; and the metabolic efficiency of folic acid is closely related to the MTHFR genotype. Furthermore, the efficacy differences of various antihypertensive drugs are also related to specific genes. Therefore, efficient and accurate detection of these key genotypes has become an essential technological pillar for improving efficacy, preventing adverse reactions, and optimizing medical resources.

[0003] Currently, various technologies are used for genotyping, but all have significant limitations when applied to large-scale clinical applications. Traditional single-site detection technologies (such as Sanger sequencing) offer high accuracy but extremely low throughput, making it difficult to meet the needs of multi-gene, multi-site detection, and are also time-consuming, labor-intensive, and costly. While gene chip technology can achieve medium-throughput detection, its detection content is fixed at the time of manufacture, making it impossible to flexibly and economically update and customize content based on new clinical guidelines or research progress. High-throughput sequencing technology can provide massive amounts of genetic information, but in targeted personalized medicine scenarios, it suffers from a serious "data overload" problem. Most of the information it generates is irrelevant to current medication decisions, instead bringing challenges such as high costs, complex data analysis, long reporting cycles, and unnecessary secondary findings. Conventional PCR technology suffers from low throughput, and when clinicians need to detect dozens of related gene loci covering multiple drugs in a single run, it is necessary to split the sample and perform a large number of independent PCR reactions, which completely negates its cost and speed advantages and significantly increases operational complexity.

[0004] In contrast, multiplex PCR-melting curve analysis offers significant advantages such as low cost, ease of operation, and rapid detection, making it highly promising for screening single or a few DNA fragments. This technique utilizes subtle differences in the melting temperatures of different DNA fragments to differentiate them, enabling clear and reliable identification of 3-5 targets within the same reaction tube.

[0005] Current pharmacogenetic typing primarily relies on invasive venous blood collection, which is dependent on professionals and suffers from low patient compliance. A new strategy for rapid and large-scale clinical gene testing is offered by collecting samples using non-invasive methods (such as oral swabs and saliva samples) combined with automated workflows. Summary of the Invention

[0006] To address the problems of low throughput and time-consuming processes in existing technologies, this invention aims to provide a primer set, kit, and application for genotyping detection of personalized medicine. This kit is based on non-invasive samples and eliminates the need for nucleic acid extraction, enabling rapid, accurate, and high-throughput detection of polymorphic sites in multiple drug metabolism genes, and is suitable for automated production lines.

[0007] To achieve the above-mentioned technical objectives, the present invention adopts the following technical solution: One objective of this invention is to provide a primer-probe set for detecting gene loci for personalized medicine, comprising: Primers and probes targeting the ApoE rs429358 gene, with sequences shown in SEQ ID NO.1~SEQ ID NO.3; Primers and probes targeting the ApoE rs7412 gene, with sequences shown in SEQ ID NO.4~SEQ ID NO.6; Primers and probes targeting the SLCO1B1 rs4149056 gene, with sequences shown in SEQ ID NO.7~SEQ ID NO.9; Primers and probes targeting the SLCO1B1 rs2306283 gene, with sequences shown in SEQ ID NO.10~SEQ ID NO.12; Primers and probes targeting the MTHFR rs1801133 gene, with sequences shown in SEQ ID NO.13~SEQ ID NO.15; Primers and probes targeting the CYP2C9 rs1057910 gene, with sequences shown in SEQ ID NO.16~SEQ ID NO.18; Primers and probes targeting the ADRB1 rs1801253 gene, with sequences shown in SEQ ID NO.19~SEQ ID NO.21; Primers and probes targeting the AGTR1 rs5186 gene, with sequences shown in SEQ ID NO.22~SEQ ID NO.24; Primers and probes targeting the CYP2D6 rs1065852 gene, with sequences shown in SEQ ID NO.25~SEQ ID NO.27; Primers and probes targeting the ACE rs1799752 gene, with sequences shown in SEQ ID NO.28~SEQ ID NO.30; Primers and probes targeting the UGT1A1 rs875347 gene, with sequences shown in SEQ ID NO.31~SEQ ID NO.33; Primers and probes targeting the UGT1A1 rs3064744 gene, with sequences shown in SEQ ID NO.34~SEQ ID NO.36; Primers and probes targeting the CYP2C19 rs4244285 gene, with sequences shown in SEQ ID NO.37~SEQ ID NO.39; Primers and probes targeting the CYP2C19 rs4986863 gene, with sequences shown in SEQ ID NO.40~SEQ ID NO.42; Primers and probes targeting the CYP2C19 rs12248560 gene, with sequences shown in SEQ ID NO.43~SEQ ID NO.45; Primers and probes targeting the ALDH2 rs671 gene, with sequences shown in SEQ ID NO.46~SEQ ID NO.48; And primers and probes targeting the CYP3A5 rs776746 gene, with sequences shown in SEQ ID NO.49~SEQ ID NO.51.

[0008] Furthermore, the 5' end of the probe is modified with any one of the fluorescent groups FAM, ROX, VIC, HEX or CY5, and the 3' end is modified with any one of the quenching groups BHQ1, BHQ2 or BHQ3.

[0009] Furthermore, when performing personalized drug gene detection, the concentration of the primers is 0.05-0.5 μM, and the concentration of the probe is 0.02-0.3 μM.

[0010] The second objective of this invention is to provide the application of the primer-probe set in the preparation of products for detecting gene loci for personalized medicine.

[0011] Furthermore, the method for detecting gene loci for personalized medicine includes the following steps: (1) Obtain oral swabs or saliva samples to be tested, mix them with nucleic acid lysis buffer, and use them directly as PCR reaction templates; (2) A PCR reaction system was constructed using the primer and probe set to perform a real-time PCR amplification reaction and obtain a melting curve; (3) Analyze the results based on the melting curve.

[0012] Furthermore, in step (2), the PCR amplification reaction program is as follows: pre-denaturation at 95℃ for 5 minutes; followed by 40 cycles of amplification, each cycle being: 95℃ for 10 seconds, 60℃ for 20 seconds, and 72℃ for 20 seconds; finally, the temperature is increased from 40℃ to 80℃, and fluorescence signals are collected every 0.06℃ for high-resolution melting curve analysis.

[0013] Furthermore, in step (3), the high-resolution melting curve analysis method is as follows: ① The sample that does not show a specific melting peak in all corresponding detection channels is a negative control; ② Positive control samples are those whose melting peaks appear in the corresponding detection channels are within the predetermined target Tm value range; ③ Determine the genotype of the locus based on the TM value of the melting curve in the fluorescence channel where the probe is located.

[0014] Furthermore, in step ③, the correspondence between genotype and Tm value is as follows:

[0016] A third objective of this invention is to provide a kit for detecting gene loci for personalized medicine, which includes the aforementioned primer and probe set.

[0017] Furthermore, the kit includes seven independent premixed PCR reaction solutions. Each reaction solution contains primers and probes targeting specific drug-related gene loci, as well as complete PCR reaction buffer and hot-start Taq DNA polymerase.

[0018] Compared with the prior art, the present invention has the following beneficial effects: This invention provides a personalized drug genotyping kit based on multiplex PCR-melting curve analysis and its applications. Through single-channel or multi-channel fluorescent PCR and melting curve analysis, high-throughput genotyping of 17 SNP loci across 12 drug-producing genes is achieved. The oral swab or saliva samples used in this invention can be directly processed with nucleic acid lysis buffer, eliminating the need for conventional DNA extraction steps, and can be used directly as templates for subsequent gene detection. The kit also offers advantages such as ease of operation, high sensitivity, and low cost, making it suitable for automated platform detection of drug metabolism gene-related genotypes and applicable for early clinical screening and precision intervention. Attached Figure Description

[0019] Figure 1 This is a detection map of the ApoE gene rs429358 (c.388T>C) site in Example 2 of the present invention.

[0020] Figure 2 This is a detection map of the ApoE gene rs7412 (c.526C>T) site in Example 2 of the present invention.

[0021] Figure 3 This is a detection map of the rs4149056 (c.521T>C) site of the SLCO1B1 gene in Example 2 of the present invention.

[0022] Figure 4 This is a detection map of the rs2306283 (c.388A>G) site of the SLCO1B1 gene in Example 2 of the present invention.

[0023] Figure 5 This is a detection map of the MTHFR gene rs1801133 (c.677C>T) site in Example 2 of the present invention.

[0024] Figure 6 This is a detection map of the rs1057910 (c.1075A>C) site of the CYP2C9 gene in Example 2 of the present invention.

[0025] Figure 7 This is a map showing the synchronous detection of the ADRB1 gene rs1801253 (c.1165G>C) and AGTR1 gene rs5186 (c.1166A>C) sites in Example 2 of the present invention.

[0026] Figure 8 This is a detection map of the rs1065852 (c.100C>T) site of the CYP2D6 gene in Example 2 of the present invention.

[0027] Figure 9This is a detection map of the ACE gene rs1799752 insertion / deletion (I / D) polymorphism in Example 2 of the present invention.

[0028] Figure 10 This is a detection map of the rs875347 (c.211G>A) site of the UGT1A1 gene in Example 2 of the present invention.

[0029] Figure 11 This is the detection map of the rs3064744 (-53(TA)6>7) site of the UGT1A1 gene in Example 2 of the present invention.

[0030] Figure 12 This is a detection map of the rs4244285 (c.681G>A) site of the CYP2C19 gene in Example 2 of the present invention.

[0031] Figure 13 This is a detection map of the rs4986863 (c.636G>A) site of the CYP2C19 gene in Example 2 of the present invention.

[0032] Figure 14 This is a detection map of the rs12248560 (c.-806C>T) site of the CYP2C19 gene in Example 2 of the present invention.

[0033] Figure 15 This is a detection map of the ALDH gene rs671 (c.1510G > A) site in Example 2 of the present invention.

[0034] Figure 16 This is a detection map of the rs776746 (c.6986A > G) site of the CYP3A5 gene in Example 2 of the present invention. Detailed Implementation

[0035] The following examples are for illustrative purposes only and are not intended to limit the scope of the invention. Any modifications or substitutions made to the methods, steps, or conditions of the invention without departing from its spirit and essence are within the scope of the invention. The reagents, products, and instruments used in the following examples are all commercially available, and the methods used in the examples, unless otherwise specified, are consistent with conventional methods.

[0036] The technical problem to be solved by this invention is to provide a primer set, kit, and application for genotyping of 12 drug-metabolizing enzyme genes for personalized medication guidance. The aim is to perform genotyping of 17 alleles from 12 drug-metabolizing enzyme genes, including CYP2C19 for clopidogrel medication guidance, ApoE and SLCO1B1 for statin medication guidance, ALDH2 for nitroglycerin medication guidance, MTHFR for folic acid medication guidance, CYP2C9, ADRB1, AGTR1, CYP2D6, and ACE for hypertension medication guidance, UGT1A1 for tumor drug toxicity guidance, and CYP3A5 for immunosuppressive drugs guidance, facilitating personalized clinical medication guidance. The oral swabs and saliva used in this invention can be directly processed with nucleic acid lysis buffer, eliminating the need for conventional DNA extraction steps, and can be used directly as templates for subsequent gene detection. This method significantly simplifies the operation process and has the advantages of being easy to operate, time-saving, and suitable for rapid on-site testing.

[0037] This invention targets 17 target sites (Table 1) of 12 genes (ApoE, SLCO1B1, MTHFR, CYP2C9, ADRB1, AGTR1, CYP2D6, ACE, UGT1A1, CYP2C19, ALDH2, and CYP3A5) based on published authoritative guidelines, databases, and professional literature from the FDA, NMPA, PharmGKB, and CPIC, according to their clinical significance and gene frequency. Primers were designed based on their related genetic information, and allele-specific amplification was performed using PCR-high resolution melting curve method to achieve genotyping.

[0038] Table 1. Details of 17 target sites in 12 drug metabolism genes.

[0039] In this invention, SEQ ID NO.1 to SEQ ID NO.51 were all synthesized by Shanghai Bailige Biotechnology Co., Ltd.

[0040] The technical solution of the present invention will be further described in detail below with reference to the embodiments.

[0041] Example 1 This invention's kit contains pre-aliquoted PCR reaction solutions in tubes. Each tube contains primer pairs, probes, and matching fluorescent dyes optimized for specific gene SNP sites. All primer sequences are designed based on the 17 key sites listed in Table 1. By adjusting product length and GC content, the melting temperature (Tm value) of each target fragment is ensured to have distinguishable differences (ΔTm ≥ 2.0℃) after multiplex PCR amplification. The reaction solutions are lyophilized and can be stably stored at room temperature. When using, only the nucleic acid template solution needs to be added for reconstitution, greatly simplifying the operation and adapting it to automated production lines.

[0042] The kit includes PCR reaction solution, positive control, negative control, and internal standard. The PCR reaction solution mainly consists of hot-start DNA Taq enzyme, amplification buffer, and sequences and probes for the target gene loci. Through the design of numerous primers and probes, and their amplification and validation on corresponding genotypes, a primer-probe combination that balances detection sensitivity and specificity based on the same PCR amplification and melting procedure was finally obtained. The specific sequences are as follows: The primer and probe sequence for ApoE rs429358 is as follows: Upstream primer SEQ ID NO.1: 5'-TGCGCAAGCTGCGTAAGC-3' Downstream primer SEQ ID NO.2: 5'-GCCCCGGCCTGGTACACTG-3' Fluorescent probe SEQ ID NO.3: ROX-5'-CCAGGCACTTCTGCAGGTCA-3'-BQ1; The ApoE rs7412 primer and probe sequence is as follows: Upstream primer SEQ ID NO.4: 5'-CACGGCTGTCCAAGGAGC-3' Downstream primer SEQ ID NO.5: 5'-AGCATGGCCTGCACCTC-3' Fluorescent probe SEQ ID NO.6: HEX-5'-ACATGGAGGACGTGTGCGGC-3'-BQ1; The primer and probe sequences for SLCO1B1 rs4149056 are as follows: Upstream primer SEQ ID NO.7: 5'-CAGTGATGTTCTTACAGTTACAGGT-3' Downstream primer SEQ ID NO.8: 5'-AACAAGTGGATAAGGTCGATGTTG-3' Fluorescent probe SEQ ID NO.9: FAM-5'-AGAAACTAATATCGATTCATCAGA-3'-BQ1; The primer and probe sequences for SLCO1B1 rs2306283 are as follows: Upstream primer SEQ ID NO.10: 5'-ATAGGTTGTTTAAAGGAATCTGGGT-3' Downstream primer SEQ ID NO.11: 5'-GCCCCAATGGTACTATGGGAGTCT-3' Fluorescent probe SEQ ID NO.12: FAM-5'-ACCCATGAACACATATATCCA-3'-BQ1; The primer and probe sequences for MTHFR rs1801133 are as follows: Upstream primer SEQ ID NO.13: 5'-GGCTGACCTGAAGCACTTGAA-3' Downstream primer SEQ ID NO.14: 5'-TTCACAAAGCGGAAGAATGTGT-3' Fluorescent probe SEQ ID NO.15: HEX-5'-TCTGCGGGAGCCGATTTCATCATC-3'-BQ1; The primer and probe sequences for CYP2C9 rs1057910 are as follows: Upstream primer SEQ ID NO.16: 5'-CCTGCATGCAAGACAGGAGC-3' Downstream primer SEQ ID NO.17: 5'-CTTCGAAAACATGGAGTTGCA-3' Fluorescent probe SEQ ID NO.18: ROX-5'-AAGGTCAAGGTATCTCTGG-3'-BQ1; The ADRB1 rs1801253 primer and probe sequence is as follows: Upstream primer SEQ ID NO.19: 5'-CGGCCTTCAACCCCATCATCT-3' Downstream primer SEQ ID NO.20: 5'-GGTCTCCGTGGGTCGCGTG-3' Fluorescent probe SEQ ID NO.21: FAM-5'-AGGCCTTCCAGGGACTGCTCTGC-3'-BQ1; The AGTR1 rs5186 primer and probe sequence is as follows: Upstream primer SEQ ID NO.22: 5'-GAGTGACATGTTCGAAACCTG-3' Downstream primer SEQ ID NO.23: 5'-AGTCGGTTCAGTCCACATAATG-3' Fluorescent probe SEQ ID NO.24: HEX-5'-CAAATGAGCCTTAGCTACT-3'-BQ1; The primer and probe sequences for CYP2D6 rs1065852 are as follows: Upstream primer SEQ ID NO.25: 5'-CATTTGGTAGTGAGGCAGGTATG-3' Downstream primer SEQ ID NO.26: 5'-TTCTGGTCCAGCCTGTGGTTTC-3' Fluorescent probe SEQ ID NO.27: FAM-5'-AGGCCTGGTGGGTAGCGTG-3'-BQ1; The primer and probe sequences for ACE rs1799752 are as follows: Upstream primer SEQ ID NO.28: 5'-CCACTCCCATCCTTTCTCCCAT-3' Downstream primer SEQ ID NO.29: 5'-CCTTAGCTCACCTCTGCTTGTAAG-3' Fluorescent probe SEQ ID NO.30: FAM-5'-AGGCGTGATACAGTCACTTTTATGTG-3'-BQ1; The UGT1A1 rs875347 primer and probe sequence is as follows: Upstream primer SEQ ID NO.31: 5'-AGTTGTCCTAGCACCTGACGC-3' Downstream primer SEQ ID NO.32: 5'-GGCACAGGGTACGTCTTCAAG-3' Fluorescent probe SEQ ID NO.33: FAM-5'-ACATCAGAGACGGAGCATTTTACA-3'-BQ1; The primer and probe sequence for UGT1A1 rs3064744 is as follows: Upstream primer SEQ ID NO.34: 5'-CACGTGACACAGTCAAACATTAAC-3' Downstream primer SEQ ID NO.35: 5'-TTTGCTCCTGCCAGAGGTTC-3' Fluorescent probe SEQ ID NO.36: FAM-5'-TGCCATATATATATATATATAAGTAGGAG-3'-BQ1; The primer and probe sequences for CYP2C19 rs4244285 are as follows: Upstream primer SEQ ID NO.37: 5'-AGCTTGGCATATTGTATCTATACCT-3' Downstream primer SEQ ID NO.38: 5'-CCAAAATATCACTTTCCATAAAAGCAAG-3' Fluorescent probe SEQ ID NO.39: HEX-5'-TGATTATTTCCCGGGAACC-3'-BHQ1; The primer and probe sequences for CYP2C19 rs4986863 are as follows: Upstream primer SEQ ID NO.40: 5'-GATCAGCAATTTCTTAACTTGATGG-3' Downstream primer SEQ ID NO.41: 5'-AAATGTACTTCAGGGCTTGGTCA-3' Fluorescent probe SEQ ID NO.42: HEX-5'-AGCACCCCCTGGATCCAG-3'-BHQ1; The primer and probe sequences for CYP2C19 rs12248560 are as follows: Upstream primer SEQ ID NO.43: 5'-CGATTTTTTTTTTCAAATTTGTGTCT-3' Downstream primer SEQ ID NO.44: 5'-GGTCTTCTGATGCCCATCGT-3' Fluorescent probe SEQ ID NO.45: ROX-5'-CATCAGAGATGCTTTGAGAACAGA-3'-BHQ1; The ALDH2 rs671 primer and probe sequences are as follows: Upstream primer SEQ ID NO.46: 5'-CAGGTCCCACACTCACAGTT-3' Downstream primer SEQ ID NO.47: 5'-TGGTGGCTACAAGATGTCGG-3' Fluorescent probe SEQ ID NO.48: ROX-5'-CAGGCATACACTGAAGTGAA-3'-BQ1; The primer and probe sequences for CYP3A5 rs776746 are as follows: Upstream primer SEQ ID NO.49: 5'-AGAGTAGAAAGCACTAC-3' Downstream primer SEQ ID NO.50: 5'-AGAGAATAAAGAGGAA-3' Fluorescent probe SEQ ID NO.51: FAM-5'-AATCAATTAAGTAACGTAGTCGT-3'-BQ1; In specific testing, PCR reaction solutions are prepared using primer and probe sets and PCR reaction reagents to detect the corresponding gene polymorphisms. Each reaction solution may include at least two fluorescent channels to detect allele types, thereby guiding clinical medication.

[0043] Example 2 The kit of the present invention contains 7 independent reaction tubes, each containing a premixed PCR reaction solution (containing primer pairs and probes corresponding to the gene to be tested, the sequences of which are shown in Example 1); the detection gene and site and the corresponding drug category for each reaction tube are shown in Table 2; the primer pairs and probe concentrations added to each reaction tube are shown in Table 2.

[0044] Table 2. Description of detection sites in reaction tubes

[0045] Using the kit of this invention and the same PCR amplification and melting curve analysis procedure, high-throughput joint genotyping of up to 12 drug-related genes and a total of 17 SNP loci can be completed in the same detection. The operation steps are as follows: Sample processing: Taking oral swab samples as an example, the samples are immersed in nucleic acid lysis buffer for cell lysis. After incubation at room temperature for 15 minutes, the lysis buffer is used directly as a template for subsequent PCR reactions. For saliva samples, 200 μL is mixed with 200 μL of nucleic acid lysis buffer and incubated at room temperature for 15 minutes. This method avoids the traditional DNA extraction steps, significantly simplifies the operation process, and is more suitable for efficient processing of batches of samples.

[0046] Sample addition: In the PCR reaction solution preparation area, select the corresponding reaction tube according to the gene site to be tested, and add template to the corresponding PCR reaction tube respectively. Specifically: add 20 μL of oral swab lysis buffer to the sample tube to be tested, add 20 μL of positive control to the positive control tube, and add 20 μL of negative control to the negative control tube.

[0047] PCR amplification and fluorescence detection: The reaction plate was placed in a real-time fluorescence PCR instrument, and a uniform amplification and detection program was run: pre-denaturation at 95℃ for 5 minutes; followed by 40 cycles of amplification (95℃ for 10 seconds, 60℃ for 20 seconds, and 72℃ for 20 seconds); finally, high-resolution melting curve analysis was performed (fluorescence signal was collected every 0.06℃ from 40℃ to 80℃). Quality control requirements: During the experiment, the negative control should not exhibit a specific melting peak in any of the corresponding detection channels; the melting peak of the positive control in the corresponding detection channel should be within the predetermined target Tm value range (see Table 3). If all the above conditions are met, the quality control of this testing process is deemed qualified; otherwise, the test should be repeated.

[0048] Results Analysis: For a sample to be considered valid, it must exhibit at least one distinct melting peak within the target Tm value range for each detection channel. If this condition is not met, the sample test fails and must be retested or the sample quality checked. Based on valid test results, genotyping can be performed according to the Tm value standards listed in Table 3.

[0049] The ApoE gene rs429358 (c.388T>C) locus shows a wild-type (TT) Tm value of 70.3±2℃, a heterozygous (TC) Tm value with a double peak of 70.3±2℃ and 76.8±2℃, and a homozygous mutant (CC) Tm value of 76.8±2℃. The rs7412 (c.526C>T) locus shows a wild-type (CC) Tm value of 66.1±2℃, a heterozygous (TC) Tm value with a double peak of 58.2±2℃ and 66.1±2℃, and a homozygous mutant (TT) Tm value of 58.2±2℃.

[0050] At the SLCO1B1 gene rs4149056 (c.521T>C) locus: the wild-type (TT) Tm value is 51.5±2℃, the heterozygous (TC) Tm value shows a double peak at 51.5±2℃ and 57.8±2℃, and the homozygous mutant (CC) Tm value is 57.8±2℃. At the rs2306283 (c.388A>G) locus: the wild-type (AA) Tm value is 50.8±2℃, the heterozygous (AG) Tm value shows a double peak at 50.8±2℃ and 56.2±2℃, and the homozygous mutant (GG) Tm value is 56.2±2℃.

[0051] MTHFR gene rs1801133 (c.677C>T) locus: wild type (CC) Tm value is 69.6±2℃, heterozygous (CT) Tm value shows a double peak of 62.8±2℃ and 69.6±2℃, homozygous mutant (TT) Tm value is 62.8±2℃.

[0052] The CYP2C9 gene rs1057910 (c.1075A>C) locus showed a wild-type (AA) Tm value of 56.0±2℃, a heterozygous (AC) Tm value with a double peak of 56.0±2℃ and 67.2±2℃, and a homozygous mutant (CC) Tm value of 67.2±2℃.

[0053] ADRB1 gene rs1801253 (c.1165G > C) site: wild type (GG) Tm value is 65.5±2℃, heterozygous (GC) Tm value shows a double peak of 65.5±2℃ and 69.2±2℃, homozygous mutant (CC) Tm value is 69.2±2℃.

[0054] The AGTR1 gene rs5186 (c.1166A>C) locus: wild type (AA) Tm value is 45.5±2℃, heterozygous (AC) Tm value shows a double peak of 45.5±2℃ and 54.5±2℃, and homozygous mutant (CC) Tm value is 54.5±2℃.

[0055] The CYP2D6 gene rs1065852 (c.100C>T) locus: wild type (CC) Tm value is 67.8±2℃, heterozygous (CT) Tm value shows a double peak of 62.7±2℃ and 67.8±2℃, and homozygous mutant (TT) Tm value is 62.7±2℃.

[0056] ACE gene rs1799752 site (I / D polymorphism): insertion homozygosity (I / I) Tm value is 65.2±2℃, heterozygosity (I / D) Tm value is 65.2±2 and 53.8±2℃, deletion homozygosity (D / D) Tm value is 53.8±2℃.

[0057] UGT1A1 gene rs875347 (c.211G>A) locus: wild-type (GG) Tm value is 64.2±2℃, heterozygous (GA) Tm value shows a double peak of 64.2±2℃ and 59.6±2℃, homozygous mutant (AA) Tm value is 59.6±2℃. rs3064744 (-53(TA)6>7) locus: (TA)6 / (TA)6 type Tm value is 52.1±2℃, (TA)6 / (TA)7 type Tm values ​​are 52.1±2℃ and 56.8±2℃, (TA)7 / (TA)7 type Tm value is 56.8±2℃.

[0058] At the CYP2C19 gene rs4244285 (c.681G>A) locus: the wild-type (GG) Tm value was 66.0±2℃, the heterozygous (GA) Tm value showed a double peak at 61.1±2℃ and 66.0±2℃, and the homozygous mutant (AA) Tm value was 61.1±2℃. At the rs4986893 (c.636G>A) locus: the wild-type (GG) Tm value was 68.3±2℃, the heterozygous (GA) Tm value showed a double peak at 62.5±2℃ and 68.3±2℃, and the homozygous mutant (AA) Tm value was 62.5±2℃. rs12248560 (c.-806C>T) site: wild type (CC) Tm value is 64.3±2℃, heterozygous (CT) Tm value shows a double peak of 56.6±2℃ and 64.3±2℃, homozygous mutant (TT) Tm value is 56.6±2℃.

[0059] The ALDH2 gene rs671 (c.1510 G> A) locus: wild type (GG) Tm value is 62.1±2℃, heterozygous (GA) Tm value shows a double peak of 62.1±2℃ and 54.8±2℃, and homozygous mutant (AA) Tm value is 54.8±2℃.

[0060] The CYP3A5 gene rs776746 (c.6986A > G) locus: wild type (AA) Tm value is 63.0±2℃, heterozygous (AG) Tm value shows a double peak of 63.0±2 and 68.1±2, and homozygous mutant (GG) Tm value is 68.1±2℃.

[0061] Table 3. Tm values ​​corresponding to genotypes

[0062] Note: If the detected peak is at the critical value between the high and low peaks, it needs to be detected again.

[0063] The embodiments described above are merely preferred embodiments of the present invention and are not intended to limit the scope of the present invention. Various modifications and improvements made by those skilled in the art to the technical solutions of the present invention without departing from the spirit of the present invention should fall within the protection scope defined by the claims of the present invention.

Claims

1. A primer probe set for detecting an individualized medication gene site, characterized in that, Among them: Primer probe for ApoE rs429358 gene, sequence as shown in SEQ ID NO. 1~SEQ ID NO. 3; Primer probe for ApoE rs7412 gene, sequence as shown in SEQ ID NO. 4~SEQ ID NO. 6; Primer probe for SLCO1B1 rs4149056 gene, sequence as shown in SEQ ID NO. 7~SEQ ID NO. 9; Primer probe for SLCO1B1 rs2306283 gene, sequence as shown in SEQ ID NO. 10~SEQ ID NO. 12; Primer probe for MTHFR rs1801133 gene, sequence as shown in SEQ ID NO. 13~SEQ ID NO. 15; Primer probe for CYP2C9 rs1057910 gene, sequence as shown in SEQ ID NO. 16~SEQ ID NO. 18; Primer probe for ADRB1 rs1801253 gene, sequence as shown in SEQ ID NO. 19~SEQ ID NO. 21; Primer probe for AGTR1 rs5186 gene, sequence as shown in SEQ ID NO. 22~SEQ ID NO. 24; Primer probe for CYP2D6 rs1065852 gene, sequence as shown in SEQ ID NO. 25~SEQ ID NO. 27; Primer probe for ACE rs1799752 gene, sequence as shown in SEQ ID NO. 28~SEQ ID NO. 30; Primer probe for UGT1A1 rs875347 gene, sequence as shown in SEQ ID NO. 31~SEQ ID NO. 33; Primer probe for UGT1A1 rs3064744 gene, sequence as shown in SEQ ID NO. 34~SEQ ID NO. 36; Primer probe for CYP2C19 rs4244285 gene, sequence as shown in SEQ ID NO. 37~SEQ ID NO. 39; Primer probe for CYP2C19 rs4986863 gene, sequence as shown in SEQ ID NO. 40~SEQ ID NO. 42; Primer probe for CYP2C19 rs12248560 gene, sequence as shown in SEQ ID NO. 43~SEQ ID NO. 45; Primer probe for ALDH2 rs671 gene, sequence as shown in SEQ ID NO. 46~SEQ ID NO. 48; And primer probe for CYP3A5 rs776746 gene, sequence as shown in SEQ ID NO. 49~SEQ ID NO.

51.

2. The primer probe set of claim 1, wherein The 5' end of the probe is modified by any one of FAM, ROX, VIC, HEX or CY5 fluorescent group, and the 3' end is modified by any one of BHQ1, BHQ2 or BHQ3 quenching group.

3. The primer probe set of claim 2, wherein When performing individualized pharmacogenetic testing, the concentration of the primer is 0.05-0.5 μM, and the concentration of the probe is 0.02-0.3 μM.

4. Use of the primer probe set according to any one of claims 1-3 in the preparation of a product for detecting individualized medication genetic sites.

5. Use according to claim 4, characterized in that, The method for detecting individualized medication genetic sites comprises the following steps: (1) Obtain the oral swab or saliva sample to be tested, mix it with a nucleic acid lysis solution, and directly use it as a PCR reaction template; (2) Construct a PCR reaction system using the primer probe set according to any one of claims 1-3 to perform a fluorescent quantitative PCR amplification reaction and obtain a melting curve; (3) Analyze the results according to the melting curve.

6. Use according to claim 5, characterized in that, In step (2), the PCR amplification reaction program is as follows: 95°C pre-denaturation for 5 minutes; then 40 cycles of amplification, each cycle: 95°C for 10 seconds, 60°C for 20 seconds, and 72°C for 20 seconds; finally, from 40°C to 80°C, collect the fluorescence signal once every 0.06°C for high-resolution melting curve analysis.

7. Use according to claim 6, characterized in that, In step (3), the high-resolution melting curve analysis method is as follows: ① The negative control product does not appear specific melting peaks in all corresponding detection channels; ② The melting peak of the positive control product appears in the corresponding detection channel within the predetermined target Tm value range; ③ The sample to be tested is judged according to the TM value of the melting curve in the fluorescence channel where the probe is located.

8. Use according to claim 7, characterized in that, In step ③, the genotype and Tm value correspondence is as follows: 。 9. A kit for detecting a personalized medicine genetic locus, characterized by, The kit comprises 7 independent premixed PCR reaction solutions, each of which contains complete PCR reaction buffer and hot-start Taq DNA polymerase in addition to primers and probes specific to certain drug-related genetic sites.

10. The kit of claim 9, wherein The kit comprises 7 independent premixed PCR reaction solutions, each of which contains complete PCR reaction buffer and hot-start Taq DNA polymerase in addition to primers and probes specific to certain drug-related genetic sites.

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