Multiplex PCR primer system, kit and method for detecting CYP2C19 gene polymorphism

Abstract

The invention discloses a multiple PCR (Polymerase Chain Reaction) primer system, a kit and a method for detecting CYP2C19 gene polymorphism. The multiple PCR primer system comprises a combination of primer pairs P1-P11, the kit comprises the multiple PCR primer system. According to the method, the multiple PCR primer system is adopted, the combination of 28 variation sites can be detected at the same time, so that 28 alleles of the CYP2C19 gene are distinguished, and the method has the advantages of being high in efficiency, easy to operate, low in cost and the like.

Description

Technical Field

[0001] The invention relates to a CYP2C19 gene SNP-specific primer multiplex PCR detection method, and in particular to a multiplex PCR primer system, a kit and a method for detecting CYP2C19 gene polymorphism, and belongs to the fields of genetic engineering and molecular biology. Background Art

[0002] The CYP2C19 gene is an important drug-metabolizing enzyme that participates in the primary metabolism of most drugs (such as amitriptyline, escitalopram, imipramine, clopidogrel, etc.), and is currently one of the most commonly tested drug genes. The polymorphism of the CYP2C19 gene is the main reason for the difference in CYP2C19 enzyme activity between individuals. Individuals carrying different CYP2C19 alleles may cause great differences in drug reactions, resulting in efficacy problems or drug toxicity (adverse reactions).

[0003] Specifically, the CYP2C19 gene is highly polymorphic, and the existing PharmVar database includes 1, 2, 3. 4,……, There are more than 30 alleles, including 39 (alleles are determined by the genotype of one or more variant sites, for specific rules, refer to the PharmVar database: https: / / www.pharmvar.org / gene / CYP2C19). Moreover, each allele involves different point mutation sites, deletions, insertions, etc., which affect the activity of CYP2C19 to varying degrees, and then affect the ability to metabolize drugs and produce different degrees of drug effects. Therefore, comprehensive multi-allelic detection and the study of the effects of CYP2C19 gene polymorphism on drug efficacy and adverse reactions can provide important scientific basis for rational clinical drug use.

[0004] At present, conventional first-generation sequencing (Sanger sequencing) detection methods for detecting CYP2C19 gene polymorphisms can generally only detect single allele-related variant sites (such as CYP2C19 2 corresponding to the variant site 19154G>A), not only the automation level is low, the detection throughput is low, but also it is mostly limited to a few alleles ( 2, 3. 17) detection, but failed to cover a more comprehensive allele range (e.g. 4. 5. 6. 7, etc.), therefore, the effects of different alleles on CYP2C19 activity and function cannot be fully evaluated.

[0005] With the development of second-generation high-throughput sequencing technology, it is possible to sequence hundreds of thousands to millions of DNA sequences in parallel at one time, greatly improving the sequencing detection throughput. Considering the purpose of detection and sequencing cost, the target region sequence is generally enriched before sequencing. Common sequence enrichment methods include chip hybridization capture and multiplex PCR amplification. Among them, chip hybridization capture can capture and enrich the entire exome or even larger target regions, but the operation process is complicated and requires reliance on specialized instruments and equipment. It is suitable for enrichment of sequences in larger target regions (such as whole exome sequencing); the experimental operation of multiplex PCR amplification is simple, and only a PCR instrument is required. It can complete the enrichment of target region sequences and library construction within a few hours, and is suitable for enrichment of relatively small target region sequences.

[0006] Therefore, it is urgent to design a primer combination with good amplification effect through multiple PCR primer design and multiple experimental optimization, so as to solve the problem of simultaneous detection of 28 variant sites of the CYP2C19 gene. Summary of the invention

[0007] In view of the above existing technical problems, the present invention discloses a multiplex PCR primer system, a kit and a method for detecting CYP2C19 gene polymorphism. By utilizing the primer combination of the multiplex PCR technology, PCR reaction and product enrichment are performed on multiple target regions at the same time, so as to achieve simultaneous detection of 28 variant sites of the CYP2C19 gene, thereby distinguishing 28 alleles.

[0008] To achieve the above object, firstly, the present invention provides a multiplex PCR primer system for detecting CYP2C19 gene polymorphism, comprising the following primer pair combinations: Primer pair P1, including upstream primer 5'-TGTTTGGAAGTTGTTTTGTTTTGCTA-3' (SEQ ID No. 1), downstream primer 5'-CACCAACAGGTGTCCTGTTCTC-3' (SEQ ID No. 2), and the amplified product covers the variant site -806C>T; Primer pair P2, including upstream primer 5'-GCTCACGGTTGTCTTAACAAGAGGA-3' (SEQ ID No. 3), downstream primer 5'-CCACTCCTCTCCCAGTGATTGGAA-3' (SEQ ID No. 4), and the amplified products covered the variant sites 1A>G, 7C>T, 10T>C, 50T>C, 55A>C, 83A>T; Primer pair P3, including upstream primer 5'-TTTCTTGCCTGGGATCTCCCTC-3' (SEQ ID No.5), downstream primer 5'-CTTGTGGAGGAGTTGAGAAAAACC-3' (SEQ ID No.6), and the amplified products covered the variable sites 12662A>G, 12711T>C, 12718A>C, 12748G>A, 12784G>A, 12802G>A; Primer pair P4, including upstream primer 5'-CCCACTTTCATCCTGGGCTGTG-3' (SEQ ID No.7), downstream primer 5'-TTCCTGAGAAACCACTTACAGTCT-3' (SEQ ID No.8), and the amplified product covers the variable sites 17869G>C, 17874G>A, 17948G>A; Primer pair P5, including upstream primer 5'-CCAGAGCTTGGCATATTGTATCTATACCTT-3' (SEQ ID No. 9), downstream primer 5'-GGAGAAAGTAAAAGAACACCAAGAATCG-3' (SEQ ID No. 10), and the amplified product covers the variant sites 19153C>T, 19154G>A; Primer pair P6, including upstream primer 5'-CCCTCGGGACTTTATTGATTGCTTC-3' (SEQ ID No. 11), downstream primer 5'-AACAGGTCAAGGAGTAATGCTTGAGA-3' (SEQ ID No. 12), and the amplified product covers the variant site 19294T>A; Primer pair P7, including upstream primer 5'-TGTCAGCTAAAGTCCAGGAAGAGAT-3' (SEQ ID No. 13), downstream primer 5'-CACGAGGTCCAGAGATACATCGA-3' (SEQ ID No. 14), and the amplified product covers the variant sites 80156G>A, 80161A>G, 80174G>A; Primer pair P8, including upstream primer 5'-TGCCCCATGCAGTGACCT-3' (SEQ ID No. 15), downstream primer 5'-TCACTGGGTGAGAGAAGTGCAT-3' (SEQ ID No. 16), and the amplified product covers the variant site 80290G>A; Primer pair P9, including upstream primer 5'-ACAGCTCAGTTCACCTATGTCTCTT-3' (SEQ ID No. 17) and downstream primer 5'-AACTACTTCATGCCTTTCTCAGCA-3' (SEQ ID No. 18), the amplified product covers the variant site 87290C>T; Primer pair P10, including upstream primer 5'-CCCTCCTATGATTCACCGAACAGT-3' (SEQ ID No. 19), downstream primer 5'-CCCGCATGGAGCTGTTTTTATTCC-3' (SEQ ID No. 20), and the amplified product covers the variant sites 90033C>T, 90060C>T, 90080C>G; Primer pair P11 includes an upstream primer 5'-CTCTGATTGACCCAAAGGACCTT-3' (SEQ ID No. 21) and a downstream primer 5'-AGATGGTCTGGCTGCTCCT-3' (SEQ ID No. 22), and the amplified product covers the variant site 90209A>C.

[0009] Secondly, the present invention provides a kit for detecting CYP2C19 gene polymorphism, comprising the multiplex PCR primer system for detecting CYP2C19 gene polymorphism.

[0010] Furthermore, the present invention provides a method for detecting CYP2C19 gene polymorphism, using the multiplex PCR primer system for detecting CYP2C19 gene polymorphism, comprising the following steps: S1. Designing primer pairs for multiple gene polymorphism sites of the CYP2C19 gene to obtain the multiplex PCR primer system for detecting CYP2C19 gene polymorphism; S2, using the nucleic acid of the sample to be tested as a template, using the multiplex PCR primer system for detecting CYP2C19 gene polymorphism obtained in S1 to perform a first round of PCR amplification, and using magnetic beads to purify the first round of PCR products to obtain purified first round PCR products; S3, performing a second round of PCR amplification on the purified first round PCR product obtained in S2, adding a sequencing adapter sequence, and purifying the second round PCR product using magnetic beads to obtain a purified second round PCR product; S4. Perform second-generation high-throughput sequencing on the purified second-round PCR products obtained in S3.

[0011] The present invention further comprises: using primer design software to first divide the CYP2C19 gene target region into multiple small target regions, and the length of the small target region is controlled within 200 bp, and then designing primer pairs for each small target region to obtain the multiplex PCR primer system for detecting CYP2C19 gene polymorphism.

[0012] In the present invention, in S2, the first round of PCR amplification reaction system includes: Enhancer buffer NB (1N) 3.5 uL, Enhancer buffer M 2.5 uL, Primer pool 5 uL, IGT-EM808 polymerase mixture 10 uL, sample + double distilled water 9 uL.

[0013] The present invention further, in said S2, the reaction conditions for the first round of PCR amplification are: first, 95°C for 3 min 30 s; then, 95°C for 20 s, 55°C for 1 min 30 s, 60°C for 1 min 30 s, 60°C for 3 min, and 18 cycles; finally, 72°C for 5 min.

[0014] In the present invention, in S3, the second round of PCR amplification reaction system includes: Enhancer buffer M 2.5uL, IGT-EM808 polymerase mixture 10uL, double distilled water 2uL, UDI Index (5μM) 2uL, and the product after the first purification 13.5uL.

[0015] The present invention further, in said S3, the reaction conditions for the second round of PCR amplification are: first, 95°C for 3 min 30 s; then, 95°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles; finally, 72°C for 5 min.

[0016] In summary, the multiplex PCR primer system and kit designed by the present invention can quickly and efficiently detect 28 variant sites of the CYP2C19 gene (-806C>T, 1A>G, 7C>T, 10T>C, 50T>C, 55A>C, 83A>T, 12662A>G, 12711T>C, 12718A>C, 12748G>A, 12784G>A, 12802G>A, 17869G>C, 17874G>A, 17948G>A, 19153C>T, 19154G>A, 19294T>A, 80156G>A, 80161A>G, 80174G>A, 80290G>A, 87290C>T, 90033C>T, 90060C>T, 90080C>G, 90209A>C) were enriched to distinguish 28 alleles ( 1, 2, 3. 4. 5. 6. 7, 8. 9. 10. 11, 12. 13. 14. 15, 16, 17, 18, twenty two, twenty four, 25, 28, 29, 32, 33, 34, 35, 39), and the detection coverage depth and uniformity can meet the requirements of second-generation sequencing for the accuracy of variant sites.

[0017] Compared with the prior art, the present invention has the following beneficial effects and technical advantages: 1. High efficiency: Since the multiplex PCR technology can use primer combinations to perform PCR reactions and product enrichment on multiple target regions at the same time, the present invention designs a set of multiplex PCR primer systems with good amplification effects through primer design and multiple experimental optimizations, which solves the problem of simultaneous detection of 28 variant sites of the CYP2C19 gene, not only improving the detection efficiency and facilitating a comprehensive evaluation of the effects of different alleles on the activity and function of CYP2C19, but also increasing the detection sensitivity, enhancing the specificity, and making the results more accurate and objective.

[0018] 2. Simple operation: The present invention only requires two rounds of PCR reactions to complete the enrichment of the target region and perform second-generation sequencing to obtain the genotype of the sample to be tested, which simplifies the operation method and reduces the labor intensity of technicians.

[0019] 3. Low cost: For application scenarios involving a large number of variant sites, the second-generation sequencing technology used in the present invention can solve the detection problem with high throughput and low cost. Compared with the first-generation sequencing, it is more cost-effective and has greater promotion advantages. BRIEF DESCRIPTION OF THE DRAWINGS

[0020] The accompanying drawings are used to provide further understanding of the present invention and constitute a part of the specification. They are used together with the embodiments of the present invention to explain the present invention and do not constitute a limitation of the present invention.

[0021] Figure 1 is a flow chart of the method of the present invention; Figure 2 Schematic diagram of the working principle of the multiplex PCR experiment in the method of the present invention. DETAILED DESCRIPTION

[0022] The preferred embodiments of the present invention are described below in conjunction with the accompanying drawings. It should be understood that the preferred embodiments described herein are only used to illustrate and explain the present invention, and are not used to limit the present invention.

[0023] Example 1: This example provides a multiplex PCR primer system for detecting CYP2C19 gene polymorphism, including one or more combinations of the following primer pairs, which are specifically described as follows.

[0024] Primer pair P1 includes upstream primer 5'-TGTTTGGAAGTTGTTTTGTTTTGCTA-3' (SEQ ID No. 1) and downstream primer 5'-CACCAACAGGTGTCCTGTTCTC-3' (SEQ ID No. 2), and the amplified product covers the variant site -806C>T.

[0025] Primer pair P2 includes upstream primer 5'-GCTCACGGTTGTCTTAACAAGAGGA-3' (SEQ ID No.3) and downstream primer 5'-CCACTCCTCTCCCAGTGATTGGAA-3' (SEQ ID No.4), and the amplified products cover the variable sites 1A>G, 7C>T, 10T>C, 50T>C, 55A>C, and 83A>T.

[0026] Primer pair P3 includes upstream primer 5'-TTTCTTGCCTGGGATCTCCCTC-3' (SEQ ID No.5) and downstream primer 5'-CTTGTGGAGGAGTTGAGAAAAACC-3' (SEQ ID No.6), and the amplified products cover the variable sites 12662A>G, 12711T>C, 12718A>C, 12748G>A, 12784G>A, and 12802G>A.

[0027] Primer pair P4 includes upstream primer 5'-CCCACTTTCATCCTGGGCTGTG-3' (SEQ ID No.7) and downstream primer 5'-TTCCTGAGAAACCACTTACAGTCT-3' (SEQ ID No.8), and the amplified products cover the variable sites 17869G>C, 17874G>A, and 17948G>A.

[0028] Primer pair P5 includes an upstream primer 5'-CCAGAGCTTGGCATATTGTATCTATACCTT-3' (SEQ ID No. 9) and a downstream primer 5'-GGAGAAAGTAAAAGAACACCAAGAATCG-3' (SEQ ID No. 10), and the amplified product covers the variable sites 19153C>T and 19154G>A.

[0029] Primer pair P6 includes an upstream primer 5'-CCCTCGGGACTTTATTGATTGCTTC-3' (SEQ ID No. 11) and a downstream primer 5'-AACAGGTCAAGGAGTAATGCTTGAGA-3' (SEQ ID No. 12), and the amplified product covers the variant site 19294T>A.

[0030] Primer pair P7 includes an upstream primer 5'-TGTCAGCTAAAGTCCAGGAAGAGAT-3' (SEQ ID No. 13) and a downstream primer 5'-CACGAGGTCCAGAGATACATCGA-3' (SEQ ID No. 14), and the amplified product covers the variable sites 80156G>A, 80161A>G, and 80174G>A.

[0031] Primer pair P8 includes an upstream primer 5'-TGCCCCATGCAGTGACCT-3' (SEQ ID No. 15) and a downstream primer 5'-TCACTGGGTGAGAGAAGTGCAT-3' (SEQ ID No. 16), and the amplified product covers the variable site 80290G>A.

[0032] Primer pair P9, including upstream primer 5'-ACAGCTCAGTTCACCTATGTCTCTT-3' (SEQ ID No. 17) and downstream primer 5'-AACTACTTCATGCCTTTCTCAGCA-3' (SEQ ID No. 18), the amplified product covers the variant site 87290C>T; Primer pair P10 includes an upstream primer 5'-CCCTCCTATGATTCACCGAACAGT-3' (SEQ ID No. 19) and a downstream primer 5'-CCCGCATGGAGCTGTTTTTATTCC-3' (SEQ ID No. 20), and the amplified product covers the variant sites 90033C>T, 90060C>T, and 90080C>G.

[0033] Primer pair P11 includes an upstream primer 5'-CTCTGATTGACCCAAAGGACCTT-3' (SEQ ID No. 21) and a downstream primer 5'-AGATGGTCTGGCTGCTCCT-3' (SEQ ID No. 22), and the amplified product covers the variant site 90209A>C.

[0034] It should be noted that a variant site may correspond to one allele or multiple alleles, and similarly, multiple variant sites may correspond to one allele. According to the data disclosed in the existing PharmVar database, the determination relationship between the detected variant site and the haplotype is shown in Table 1 below.

[0035] Table 1: Correspondence table of the determination relationship between detected variant sites and haplotypes As can be seen from Table 1, the relationship between each primer pair and each allele in the multiplex PCR primer system of the present invention is as follows.

[0036] Primer pair P1 is used to detect the CYP2C19 allele 4. CYP2C19 17.

[0037] Primer pair P2 is used to detect the CYP2C19 allele 4. CYP2C19 14. CYP2C19 15.CYP2C19 28, CYP2C19 29, CYP2C19 34, CYP2C19 35, CYP2C19 39.

[0038] Primer pair P3 is used to detect CYP2C19 alleles 2. CYP2C19 6. CYP2C19 8. CYP2C19 9. CYP2C19 11. CYP2C19 35, CYP2C19 39.

[0039] Primer pair P4 is used to detect CYP2C19 alleles 3. CYP2C19 22, CYP2C19 33.

[0040] Primer pair P5 is used to detect CYP2C19 alleles 2. CYP2C19 10.

[0041] Primer pair P6 is used to detect CYP2C19 alleles 7.

[0042] Primer pair P7 is used to detect CYP2C19 alleles 16, CYP2C19 18, CYP2C19 24, CYP2C19 34, CYP2C19 38.

[0043] Primer pair P8 is used to detect CYP2C19 alleles 28.

[0044] Primer pair P9 is used to detect CYP2C19 alleles 13.

[0045] Primer pair P10 is used to detect CYP2C19 alleles 5. CYP2C19 16, CYP2C19 25.

[0046] Primer pair P11 is used to detect the CYP2C19 allele 12.

[0047] Furthermore, when all involved variant sites are wild type, the allele CYP2C19 is detected. 1. That is, there is no other haploid in Table 1, such as 2. 3.…… 39, etc., it is naturally judged that the haploid 1.

[0048] In specific implementation, the multiplex PCR primer system for detecting CYP2C19 gene polymorphism provided in this embodiment includes one or more combinations of the above primer pairs (primer pair P1, primer pair P2, primer pair P3, primer pair P4, primer pair P5, primer pair P6, primer pair P7, primer pair P8, primer pair P9, primer pair P10, primer pair P11), which can detect 28 alleles (CYP2C19 1. CYP2C19 2. CYP2C19 3. CYP2C19 4. CYP2C19 5. CYP2C19 6. CYP2C19 7. CYP2C19 8. CYP2C19 9. CYP2C19 10. CYP2C19 11. CYP2C19 12. CYP2C19 13. CYP2C19 14. CYP2C19 15.CYP2C19 16, CYP2C19 17, CYP2C19 18, CYP2C19 22, CYP2C19 24, CYP2C19 25, CYP2C19 28, CYP2C19 29, CYP2C19 32, CYP2C19 33, CYP2C19 34, CYP2C19 35, CYP2C19 39) or a combination of one or more of the following.

[0049] Of course, since different primer pair combinations can distinguish different haploid combinations, the multiplex PCR primer system provided in this embodiment can detect different allele combinations, all of which are within the scope of protection of the present invention. If the multiplex PCR primer system includes all of the above primer pairs, the 28 variant sites of the CYP2C19 gene can be detected simultaneously, which not only improves the efficiency of the detection, but also comprehensively evaluates the effects of different alleles on the activity and function of CYP2C19.

[0050] Example 2: This example provides a kit for detecting CYP2C19 gene polymorphism, including the multiplex PCR primer system for detecting CYP2C19 gene polymorphism described in Example 1.

[0051] In a specific implementation, the kit for detecting CYP2C19 gene polymorphism further includes DNA polymerase, deoxynucleotide triphosphate (dNTP), polyethylene glycol, dimethyl sulfoxide, magnesium chloride, sodium chloride, water, etc.

[0052] As can be seen from Example 1, the multiplex PCR primers for detecting CYP2C19 gene polymorphism of the present invention can detect 28 variant sites (-806C>T, 1A>G, 7C>T, 10T>C, 50T>C, 55A>C, 83A>T, 12662A>G, 12711T>C, 12718A>C, 12748G>A, 12784G>A, 12802G>A, 17869G>C, 17874G>A, 17948G>A, 19153C>T, 19154G>A, 19294T>A, 80156G>A, 80161A>G, 80174G>A, 80290G>A, 87290C>T, 90033C>T, 90060C>T, 90080C>G, 90209A>C) can be detected simultaneously, so the polymorphism detection kit of the present invention can detect 28 alleles (CYP2C19 1. CYP2C19 2. CYP2C19 3. CYP2C19 4. CYP2C19 5. CYP2C19 6. CYP2C19 7. CYP2C19 8. CYP2C19 9. CYP2C19 10. CYP2C19 11. CYP2C19 12. CYP2C19 13. CYP2C19 14. CYP2C19 15.CYP2C19 16, CYP2C19 17, CYP2C19 18, CYP2C19 22, CYP2C19 24, CYP2C19 25, CYP2C19 28, CYP2C19 29, CYP2C19 32, CYP2C19 33, CYP2C19 34, CYP2C19 35, CYP2C19 39) are tested simultaneously in combination with one or more of the above.

[0053] Therefore, the kit provided in this embodiment achieves the effect of detecting CYP2C19 gene polymorphism and solves the problem of simultaneously detecting 28 variant sites of CYP2C19 gene. This not only improves the efficiency of detection, but also has higher detection sensitivity, stronger specificity, and more accurate and objective results.

[0054] Example 3: Figure 1 As shown, this embodiment provides a method for detecting CYP2C19 gene polymorphism, using the multiplex PCR primer system for detecting CYP2C19 gene polymorphism described in Example 1, and the kit for detecting CYP2C19 gene polymorphism described in Example 2, comprising the following steps: S1. Design primer pairs for multiple gene polymorphism sites of the CYP2C19 gene to obtain the multiplex PCR primer system for detecting CYP2C19 gene polymorphism as described in Example 1.

[0055] In the specific implementation, conventional primer design software was used to design primer pairs for the target region of the CYP2C19 gene: the target region of the CYP2C19 gene was first divided into multiple small target regions, and the length of the small target regions was controlled within 200 bp. Then, primer pairs were designed for each small target region, and the following factors were considered: (1) the amplification efficiency of the primers for a single fragment; (2) whether there was mutual interference between primers, such as the formation of primer dimers, which reduced the amplification efficiency; (3) the balance of amplification efficiency between different primers in the same reaction system: because multiple pairs of primers were placed under the same reaction conditions (PCR temperature setting, etc.), it was necessary to ensure that each pair of primers could exert a certain amplification efficiency; at the same time, the amplification efficiency between different primers would not differ greatly and had a certain uniformity, otherwise the data utilization rate in the subsequent sequencing link would be affected. The final multiplex PCR primer system is shown in Table 2, and a primer pool was generated for subsequent use.

[0056] Table 2: List of primer pairs for CYP2C19 gene amplification It should be noted that the combination of primer pair P1 to primer pair P11 shown in Table 2 is the best solution for the multiplex PCR primer system of the present invention, covering the most variable sites of the CYP2C19 gene, and can provide the highest haplotype resolution, that is, can distinguish 28 alleles at the same time. If the multiplex PCR primer system of the present invention only includes some of the primer pairs shown in Table 2, only some haplotypes can be distinguished, which is also within the scope of protection of the present invention.

[0057] S2, such as Figure 2 As shown, the total DNA (gDNA) of the sample to be tested is used as a template, the multiplex PCR primer system obtained in S1 is used to perform the first round of PCR amplification, and the first round of PCR products are purified using magnetic beads.

[0058] In the specific implementation, S2-1, using the total DNA (gDNA) of the sample to be tested as a template, a first round of PCR amplification is performed using a multiplex PCR primer system to obtain a first round of PCR products.

[0059] The reaction system for the first round of PCR amplification includes: Enhancer buffer NB (1N) 3.5uL, Enhancer buffer M 2.5uL, Primer pool 5uL, IGT-EM808polymerase mixture 10uL, sample + double distilled water 9uL. In addition, the general sample starting amount is required to be no less than 40ng / reaction tube.

[0060] The reaction conditions of the first round of PCR amplification were: first, 95°C for 3 min 30 s; then, 95°C for 20 s, 55°C for 1 min 30 s, 60°C for 1 min 30 s, 60°C for 3 min, and 18 cycles, and finally, 72°C for 5 min. The above are the optimal reaction temperatures and reaction times.

[0061] S2-2. Purifying the first-round PCR products using magnetic beads can effectively remove contamination such as oligonucleotides, primer dimers, salts, and proteins in the first-round PCR products to obtain purified first-round PCR products.

[0062] S3, such as Figure 2 As shown, the purified first-round PCR product obtained by S2 is subjected to a second-round PCR amplification, sequencing adapter sequences are added to both ends of the DNA fragment, and the second-round PCR product is purified using magnetic beads.

[0063] In specific implementation, S3-1, the product after the first purification is subjected to a second round of PCR amplification, and sequencing adapter sequences are added to both ends of the DNA fragment to obtain the second round of PCR products.

[0064] The reaction system for the second round of PCR amplification included: Enhancer buffer M 2.5uL, IGT-EM808polymerase mixture 10uL, double distilled water 2uL, UDI Index (5μM) 2uL, and the product after the first purification 13.5uL.

[0065] The reaction conditions of the second round of PCR amplification are: first, 95°C for 3 min 30 s; then, 95°C for 20 s, 58°C for 1 min, 72°C for 30 s, 9 cycles; finally, 72°C for 5 min. And the above are the optimal reaction temperature and reaction time.

[0066] S3-2. Purifying the second-round PCR products using magnetic beads can effectively remove contamination such as oligonucleotides, primer dimers, salts, and proteins in the second-round PCR products to obtain purified second-round PCR products.

[0067] S4, such as Figure 2 As shown, the purified second-round PCR products obtained in S3 are used to construct a sequencing library, and second-generation high-throughput sequencing is performed to obtain the genotype of the sample to be tested.

[0068] During specific implementation, the normal concentration range of the sequencing library is 20-50 ng / uL.

[0069] Furthermore, the second-generation high-throughput sequencing refers to a second-generation sequencing method based on the MGI or Illumina platform.

[0070] Example 4: In order to verify that the multiplex PCR primer system designed in the present invention can quickly and efficiently enrich the 28 polymorphic site-related regions of the CYP2C19 gene, and the detection coverage depth and uniformity can meet the requirements of the second-generation sequencing for the accuracy of the variant sites, the following detection experiment steps are followed to detect different samples 1, sample 2, and sample 3, respectively, as described below.

[0071] A1. Collection and extraction of sample genomic DNA: First, use the ORAcollect DNA OCR-100 (DNAGenotek) saliva collector to collect saliva samples of sample 1, sample 2, and sample 3 according to the instructions; then use the Magen-MD5111 DNA extraction kit to extract the total DNA of sample 1, sample 2, and sample 3, respectively.

[0072] A2. Use Qubit 4 Fluorometer to measure the DNA concentration of sample 1, sample 2, and sample 3. The DNA concentrations of sample 1, sample 2, and sample 3 are 33.2 ng / uL, 37.6 ng / uL, and 39.2 ng / uL, respectively, which meet the requirements of multiplex PCR experiments.

[0073] A3. First, the multiplex PCR primer system including primer pair P1 to primer pair P11 of Example 1 was added to sample 1, sample 2, and sample 3, respectively. Then, the first round of PCR amplification was performed on sample 1, sample 2, and sample 3 using an Applied Biosystems Veriti PCR instrument to enrich the target region sequence, and the first round PCR products of sample 1, sample 2, and sample 3 were obtained, respectively.

[0074] During the specific implementation, the PCR reagent used was MultipSeq® Library Prep Kit (M61012) produced by Agenix.

[0075] The reaction system for the first round of PCR amplification was 30uL, including: Enhancer buffer NB (1N) 3.5uL, Enhancer buffer M 2.5uL, Primer pool 5uL, IGT-EM808polymerase mixture 10uL, sample + double distilled water 9uL.

[0076] The reaction conditions for the first round of PCR amplification were set as: first 95°C for 3 min 30 s; then 18 cycles (95°C for 20 s, 55°C for 1 min 30 s, 60°C for 1 min 30 s, and 60°C for 3 min), and finally 72°C for 5 min.

[0077] A4. Use a magnetic stand and purification magnetic beads (IGT Pure Beads) to purify the first-round PCR products of sample 1, sample 2, and sample 3, respectively, to obtain the purified first-round PCR products of sample 1, sample 2, and sample 3, respectively.

[0078] A5. Use Applied Biosystems Veriti PCR instrument to perform second-round PCR amplification on the purified first-round PCR products of sample 1, sample 2, and sample 3, respectively, for adapter sequence PCR reaction to obtain second-round PCR products of sample 1, sample 2, and sample 3, respectively.

[0079] During the specific implementation, the PCR reagent used was MultipSeq® Library Prep Kit (M61012) produced by Agenix.

[0080] Among them, the reaction system of the second round of PCR amplification is 30uL, including: Enhancer buffer M 2.5uL, IGT-EM808 polymerase mixture 10uL, double distilled water 2uL, UDI Index (5μM) 2uL, and the purified first round PCR product 13.5uL.

[0081] The reaction conditions for the second round of PCR amplification were set as: first 95°C for 3 min 30 s; then 9 cycles (95°C for 20 s, 58°C for 1 min, and 72°C for 30 s); and finally 72°C for 5 min.

[0082] In addition, the UDI Index adapter sequence used MultipSeq® UDI Primer 1-96 (5 μM each, for MGI, plate; M70132).

[0083] A6. Use a magnetic stand and purification magnetic beads (IGT Pure Beads) to purify the second-round PCR products of sample 1, sample 2, and sample 3, respectively, to obtain the purified second-round PCR products of sample 1, sample 2, and sample 3, respectively.

[0084] A7. Use Qubit 4 Fluorometer to measure the DNA concentration of the second-round PCR products after purification of sample 1, sample 2, and sample 3. The DNA concentrations of sample 1, sample 2, and sample 3 were 28.8 ng / uL, 33.3 ng / uL, and 32.2 ng / uL, respectively, which all meet the requirements of the second-generation sequencing experiment.

[0085] A8. Use the MGI platform (DNBSEQ-G99) method to perform next-generation sequencing on the purified second-round PCR products of sample 1, sample 2, and sample 3, respectively, and perform data analysis.

[0086] In the specific implementation, first, after the detection experiments were carried out on three different samples according to the above steps, the sequencing depths of each region in the three detection experiments are shown in Table 3. From the results of the three experiments, it can be seen that when the multiplex PCR primer system including primer pair P1 to primer pair P11 in Example 1 is used, the depth of all target regions is at least 124x when the average sequencing depth is about 500x, and the depth can meet the requirements of the second-generation sequencing bioinformatics analysis for the accuracy of detecting variant sites (sequencing depth is not less than 30x).

[0087] Table 3: Average sequencing depth of each sample in each primer pair region in the test results of sample 1, sample 2, and sample 3.

[0088] Secondly, the uniformity of the target region captured by the multiplex PCR method was calculated, and the proportion of bases in the target region greater than 0.2 times the average sequencing depth was used as an evaluation index, and the proportions of sample 1, sample 2, and sample 3 were 95.83%, 95.83%, and 95.83%, respectively. It can be seen that the amplification uniformity of the combination of primer pair P1 to primer pair P11 in Example 1 is relatively high, and the genotype of each site can be well detected, thereby producing high-quality bioinformatics analysis data.

[0089] In summary, the multiplex PCR primer system and detection method of the present invention can efficiently and reliably perform enrichment and high-throughput sequencing detection on the CYP2C19 gene, and cover a more comprehensive range of alleles, especially being able to simultaneously detect 28 alleles, thereby improving the detection efficiency and comprehensively evaluating the effects of different alleles on the activity and function of CYP2C19.

[0090] Finally, it should be noted that although the present invention has provided specific embodiments for description, these embodiments are only used to explain the present invention and do not limit the invention. In any one or more embodiments or examples, the specific features, structures, materials or characteristics described may be combined in a suitable manner. After reading this specification, those skilled in the art may modify, replace and modify the embodiments as needed without violating the principles and purpose of the present invention. Although these changes do not make creative contributions, they are still protected by patent law as long as they are within the scope of the claims of the present invention.

Claims

1. A multiplex PCR primer system for detecting CYP2C19 gene polymorphism, characterized in that: Combinations of primer pairs include: Primer pair P1, including upstream primer 5'-TGTTTGGAAGTTGTTTTGTTTTGCTA-3' (SEQ ID No. 1), downstream primer 5'-CACCAACAGGTGTCCTGTTCTC-3' (SEQ ID No. 2), and the amplified product covers the variant site -806C>T; Primer pair P2, including upstream primer 5'-GCTCACGGTTGTCTTAACAAGAGGA-3' (SEQ ID No. 3), downstream primer 5'-CCACTCCTCTCCCAGTGATTGGAA-3' (SEQ ID No. 4), and the amplified products covered the variant sites 1A>G, 7C>T, 10T>C, 50T>C, 55A>C, 83A>T; Primer pair P3, including upstream primer 5'-TTTCTTGCCTGGGATCTCCCTC-3' (SEQ ID No.5), downstream primer 5'-CTTGTGGAGGAGTTGAGAAAAACC-3' (SEQ ID No.6), and the amplified products covered the variable sites 12662A>G, 12711T>C, 12718A>C, 12748G>A, 12784G>A, 12802G>A; Primer pair P4, including upstream primer 5'-CCCACTTTCATCCTGGGCTGTG-3' (SEQ ID No.7), downstream primer 5'-TTCCTGAGAAACCACTTACAGTCT-3' (SEQ ID No.8), and the amplified product covers the variable sites 17869G>C, 17874G>A, 17948G>A; Primer pair P5, including upstream primer 5'-CCAGAGCTTGGCATATTGTATCTATACCTT-3' (SEQ ID No. 9), downstream primer 5'-GGAGAAAGTAAAAGAACACCAAGAATCG-3' (SEQ ID No. 10), and the amplified product covers the variant sites 19153C>T, 19154G>A; Primer pair P6, including upstream primer 5'-CCCTCGGGACTTTATTGATTGCTTC-3' (SEQ ID No. 11), downstream primer 5'-AACAGGTCAAGGAGTAATGCTTGAGA-3' (SEQ ID No. 12), and the amplified product covers the variant site 19294T>A; Primer pair P7, including upstream primer 5'-TGTCAGCTAAAGTCCAGGAAGAGAT-3' (SEQ ID No. 13), downstream primer 5'-CACGAGGTCCAGAGATACATCGA-3' (SEQ ID No. 14), and the amplified product covers the variant sites 80156G>A, 80161A>G, 80174G>A; Primer pair P8, including upstream primer 5'-TGCCCCATGCAGTGACCT-3' (SEQ ID No. 15), downstream primer 5'-TCACTGGGTGAGAGAAGTGCAT-3' (SEQ ID No. 16), and the amplified product covers the variant site 80290G>A; Primer pair P9, including upstream primer 5'-ACAGCTCAGTTCACCTATGTCTCTT-3' (SEQ ID No. 17) and downstream primer 5'-AACTACTTCATGCCTTTCTCAGCA-3' (SEQ ID No. 18), the amplified product covers the variant site 87290C>T; Primer pair P10, including upstream primer 5'-CCCTCCTATGATTCACCGAACAGT-3' (SEQ ID No. 19), downstream primer 5'-CCCGCATGGAGCTGTTTTTATTCC-3' (SEQ ID No. 20), and the amplified product covers the variant sites 90033C>T, 90060C>T, 90080C>G; Primer pair P11 includes an upstream primer 5'-CTCTGATTGACCCAAAGGACCTT-3' (SEQ ID No. 21) and a downstream primer 5'-AGATGGTCTGGCTGCTCCT-3' (SEQ ID No. 22), and the amplified product covers the variant site 90209A>C.

2. A kit for detecting CYP2C19 gene polymorphism, characterized in that: It comprises the multiplex PCR primer system for detecting CYP2C19 gene polymorphism as claimed in claim 1.

3. A method for detecting CYP2C19 gene polymorphism, characterized in that: The multiplex PCR primer system for detecting CYP2C19 gene polymorphism as claimed in claim 1 comprises the following steps: S1. Designing primer pairs for multiple gene polymorphism sites of the CYP2C19 gene to obtain the multiplex PCR primer system for detecting CYP2C19 gene polymorphism; S2, using the total DNA of the sample to be tested as a template, using the multiplex PCR primer system for detecting CYP2C19 gene polymorphism obtained in S1 to perform the first round of PCR amplification, and using magnetic beads to purify the first round of PCR products to obtain purified first round PCR products; S3, performing a second round of PCR amplification on the purified first round PCR product obtained in S2, adding sequencing adapter sequences to both ends of the DNA fragment, and purifying the second round PCR product using magnetic beads to obtain a purified second round PCR product; S4. Perform second-generation high-throughput sequencing on the purified second-round PCR products obtained in S3 to obtain the genotype of the sample to be tested.

4. A method for detecting CYP2C19 gene polymorphism according to claim 3, characterized in that: The S1 comprises: using primer design software, first dividing the CYP2C19 gene target region into multiple small target regions, and controlling the length of the small target regions within 200 bp, and then designing primer pairs for each small target region to obtain the multiplex PCR primer system for detecting CYP2C19 gene polymorphism.

5. A method for detecting CYP2C19 gene polymorphism according to claim 3, characterized in that: In S2, the reaction system for the first round of PCR amplification includes: Enhancer buffer NB (1N) 3.5uL, Enhancer buffer M 2.5uL, Primer pool 5uL, IGT-EM808 polymerase mixture 10uL, sample + double distilled water 9uL.

6. A method for detecting CYP2C19 gene polymorphism according to claim 3 or 5, characterized in that: In S2, the reaction conditions for the first round of PCR amplification are: first, 95°C for 3 min 30 s; then, 95°C for 20 s, 55°C for 1 min 30 s, 60°C for 1 min 30 s, 60°C for 3 min, and 18 cycles; finally, 72°C for 5 min.

7. A method for detecting CYP2C19 gene polymorphism according to claim 3, characterized in that: In S3, the reaction system for the second round of PCR amplification includes: Enhancer buffer M 2.5uL, IGT-EM808 polymerase mixture 10uL, double distilled water 2uL, UDI Index (5μM) 2uL, and the product after the first purification 13.5uL.

8. A method for detecting CYP2C19 gene polymorphism according to claim 3 or 7, characterized in that: In S3, the reaction conditions for the second round of PCR amplification are: first, 95°C for 3 min and 30 s; then, 95°C for 20 s, 58°C for 1 min, and 72°C for 30 s, for 9 cycles; and finally, 72°C for 5 min.

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