Composite amplification system, detection method and kit for genotyping of anesthetic

Abstract

The invention provides a composite amplification system, a kit and a detection method for polymorphism detection of anesthetic related genes, and relates to the technical field of drug gene detection. According to the invention, a series of specific primer compositions as shown in SEQ ID NO.1-133 and a multiplex amplification system thereof are designed aiming at 44 SNP loci corresponding to 24 drugs in 7 classes; the kit obtained by utilizing the primer composition or the composite amplification system has very high sensitivity to 44 SNP sites related to anesthetic, and the detection result is high in accuracy and can be kept consistent with a gold standard; meanwhile, the kit and the detection method provided by the invention support direct filling of blood or a blood card, do not need to pre-treat a to-be-detected sample, can directly obtain a detection result, are simple in operation steps and short in time consumption, can greatly reduce the cost, and have the advantages of high sensitivity, high resolution, high flux and the like. By utilizing the method, the genetic typing of the patient on the related anesthetic can be quickly and accurately obtained, the evaluation of the patient on the adverse reaction and toxicity early warning, curative effect evaluation and the like on the anesthetic is realized, more gene reference information of the patient is provided for clinicians, and an effective evaluation index is provided for individualized use of the anesthetic.

Description

Technical Field

[0001] The present invention relates to the technical field of drug gene detection, and in particular to a composite amplification system, a detection method and a kit for gene typing of anesthetic drugs. Background Art

[0002] Modern anesthesiology began in 1846. In the clinical application of anesthetics, when different patients use the same type and the same dose of anesthetics, there are obvious individual differences in the time of loss of consciousness, awakening time, depth of anesthesia, and the incidence of adverse reactions. Many studies have shown that genetic factors are an important cause of differences in pharmacokinetic and pharmacodynamic responses between individuals. Pharmacogenomics is a discipline that studies the polymorphism of genes related to drug metabolism and pharmacodynamics and individual differences in drug response. It aims to find drug metabolizing enzymes, drug transporters, and targets related to individual differences in drug response. Anesthetic pharmacogenomics research can provide an important basis for the individualized use of anesthetics and the prevention of adverse reactions, and improve the safety of anesthetics during surgery.

[0003] In recent years, a variety of new methods and technologies for detecting gene polymorphisms have appeared on the market, including denaturing high pressure liquid chromatography (DHPLC), primer extension combined with time-of-flight mass spectrometry (MALDI-TOFMS), dynamic allele-specific hybridization, gene chip method, TaqMan probe technology, and pyrophosphate sequencing technology, etc. These methods and technologies have significantly improved in sensitivity and throughput, making them increasingly widely used in the field of genetic testing.

[0004] Although these technologies can effectively detect single nucleotide polymorphisms (SNPs), they also have some limitations in practical applications. For example, the operation process of some methods is complicated and requires restriction endonuclease digestion; some require two-step PCR amplification reactions. In addition, although some new technologies have the advantages of high throughput and easy automation, they usually require expensive instruments and equipment. Some other technologies have shortcomings in repeatability, result interpretation, number of detection sites, throughput, detection cycle and cost, such as poor repeatability, difficult results, few detection sites, low detection throughput, long detection cycle and high detection cost. Summary of the invention

[0005] 1. Technical issues to be resolved

[0006] In view of the deficiencies in the prior art, the present invention provides a composite amplification system, a kit and a detection method for detecting gene polymorphisms related to anesthetic drugs with high throughput, strong specificity, high sensitivity, low cost and simple operation.

[0007] (II) Technical solution

[0008] To achieve the above objectives, the present invention is implemented through the following technical solutions:

[0009] In a first aspect, the present invention provides a primer composition for detecting gene polymorphisms related to anesthetic medication, wherein the primer composition comprises 133 primers, and the sequences thereof are shown in SEQ ID NO.1-133.

[0010] The above primer set was obtained after extensive screening and analysis, and primers were designed for 44 SNP sites corresponding to 24 drugs in 7 categories commonly used in anesthesiology. The SNP sites are as follows:

[0011]

[0012] Furthermore, the primers in the primer set further include one of fluorescent group modification, phosphorylation modification, thiophosphorylation modification, locked nucleic acid modification or peptide nucleic acid modification.

[0013] In a second aspect, the present invention provides a composite amplification system for detecting gene polymorphisms related to anesthetic drugs, wherein the composite amplification system comprises a primer combination shown in sequence SEQ ID NO.1-133, and the composite amplification system is a system combining multiple fluorescent ARMS PCR with capillary electrophoresis detection.

[0014] Furthermore, in this system, the detection adopts capillary electrophoresis detection, and the amplification adopts multiple allele-specific PCR amplification.

[0015] In a third aspect, the present invention further provides a kit for detecting gene polymorphisms related to anesthetic medication, wherein the kit comprises a primer combination or a composite amplification system shown in the sequence SEQ ID NO.1-133.

[0016] Furthermore, the detection kit adopts the method of allele-specific PCR (ASPCR) combined with quantitative fluorescent PCR (QF-PCR) to amplify the target site, and detects the amplified product by capillary electrophoresis to complete the detection of the target site typing; three primers are set for each detection site, and a specific primer of different lengths and a fluorescently labeled downstream primer are set for the two typings respectively; each specific primer can only bind to and amplify the DNA template of the corresponding genotype; after completing PCR amplification and capillary electrophoresis detection, the presence or absence of amplification products with specific fluorescent labels and specific lengths can be used to determine whether a specific genotype exists at a specific site of the sample. Generally, the target fragment amplified by the wild-type primer will be 4bp smaller than the fragment amplified by the mutant primer.

[0017] Furthermore, the kit also includes hot-start DNA Taq enzyme and 2×PCR amplification buffer.

[0018] Furthermore, the kit also includes quality control products and related reagents required for capillary detection.

[0019] In a fourth aspect, the present invention provides a method for detecting gene polymorphisms related to anesthetic drugs, the method comprising: performing PCR amplification and capillary electrophoresis detection on the test sample using the above-mentioned primer composition or the above-mentioned composite amplification system or the above-mentioned kit, and then determining whether a specific genotype exists at a specific site of the sample by the presence or absence of an amplification product with a specific fluorescent marker and a specific length.

[0020] (III) Beneficial effects

[0021] The present invention provides a composite amplification system, a kit and a detection method for detecting gene polymorphisms related to anesthetic drugs, which have the following advantages over the prior art:

[0022] 1. Based on the principle of allele-specific PCR, the present invention first preliminarily designs specific primer sequences for 44 SNP sites corresponding to 24 drugs in 7 categories. In order to coordinate amplification efficiency, improve product peak shape, and improve detection efficiency, we further make a series of specific changes or modifications to all primers, and finally obtain a primer composition shown in the sequence SEQ ID NO.1-133; the primer composition covers a comprehensive range of sites and can realize multiple specific detection of multiple SNP sites.

[0023] 2. The kit provided by the present invention has high detection accuracy for 44 SNP sites related to anesthetic drugs and can keep consistent with the gold standard; at the same time, the kit provided by the present invention supports direct charging of blood or blood cards, and does not require pretreatment of the test samples, and can directly obtain the test results, with simple operation steps.

[0024] 3. The detection method provided by the present invention is simple to operate, short in time, can greatly reduce costs, and has the advantages of high sensitivity, high resolution, and high throughput.

[0025] 4. The composite amplification system, kit and detection method for detecting anesthetic-related gene polymorphism provided by the present invention can perform multiple amplification detection on 44 SNP sites corresponding to 24 drugs in seven categories commonly used in the anesthesiology department, thereby realizing the patient's evaluation of the toxicity, malignant symptom warning, efficacy evaluation, etc. of anesthetic drugs, providing clinicians with more patient genetic information and providing effective evaluation indicators for the individualized use of anesthetic drugs. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] Figure 1Screening results of primers for CYP2D6*10 (rs1065852) site.

[0027] Figure 2 The first group of primer combinations for constructing the amplification system were optimized (from top to bottom, they are combination 1, combination 2, and combination 3).

[0028] Figure 3 The kit amplifies the clinical sample of Example 3 and performs capillary electrophoresis detection. DETAILED DESCRIPTION

[0029] In order to make the purpose, technical solution and advantages of the embodiments of the present invention clearer, the technical solution in the embodiments of the present invention will be clearly and completely described in combination with the embodiments of the present invention. Obviously, the described embodiments are part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0030] Example 1

[0031] Screening and identification of gene loci related to anesthetic drugs

[0032] This example comprehensively screens the targets that affect the efficacy of antidepressant drugs based on the expert guidelines for drug metabolizing enzymes and drug target gene detection technology guidelines (trial) formulated by the National Health and Family Planning Commission's Individualized Medicine Testing Technology Expert Committee, CPIC, diagnosis and treatment guidelines, FDA drug instructions, and clinical evidence levels in the pharmGKB database.

[0033] This example initially screened 24 anesthesia-related drugs in 7 categories. The drug classification and drug target are included in the following table:

[0034]

[0035]

[0036]

[0037] Furthermore, considering the characteristics of the gene sequences at each site, the size arrangement of the sites in the system, and the difficulty of system optimization, 44 SNP sites were finally selected as targets for subsequent testing. The genes and information are as follows:

[0038]

[0039]

[0040] Example 2

[0041] Construction and optimization of a detection system for gene polymorphisms related to anesthetic drugs

[0042] (1) Preliminary design of primers for selected sites

[0043] According to the principle of allele-specific PCR, each specific primer can only bind to and amplify the DNA template of the corresponding genotype. The primer sequences are preliminarily designed as follows:

[0044] CACNA1S gene rs772226819 site:

[0045] Forward wild-type primer: 5'-TCCGAGTGCTCAGACCCCTCC-3',

[0046] Forward mutant primer: 5'-GCCTTCCGAGTGCTCAGACCCCTCT-3'.

[0047] Reverse common primer: 5′-ACCCAGAACTGGGGGACAAG-3′;

[0048] CYP2D6 gene rs1065852 site:

[0049] Forward wild-type primer: 5'-GTGGCAGGGGGCCTGGTGG-3',

[0050] Forward mutant primer: 5'-GGCAGTGGCAGGGGGCCTGGTGA-3',

[0051] Reverse common primer: 5′-ACCCATTTGGTAGTGAGGCAGGT-3′;

[0052] G6PD gene rs72554664 site:

[0053] Forward wild-type primer: 5'-GCAGCAGTGGGGTGAAAATAC-3',

[0054] Forward mutant primer: 5'-TGGTGCACCAGTGGGGTGAAAATAT-3',

[0055] Reverse common primer: 5′-AGGGAGCCAGATGCACTTCGT-3′;

[0056] BChE gene rs1803274 site:

[0057] Forward wild-type primer: 5'-TGCTTTCCACTCCCATTCTGC-3',

[0058] Forward mutant primer: 5'-ATCCTGCTTTCCACTCCCATTCTGT-3',

[0059] Reverse common primer: 5′-TCAGGCAAAGCGAGCTAATAACAA-3′;

[0060] FAAH gene rs4141964 site:

[0061] Forward wild-type primer: 5'-ACCAAAGTAAGTCTGCACCTCCAGA-3',

[0062] Forward mutant primer: 5'-AGACACCAAAGTAAGTCTGCACCTCCAGG-3',

[0063] Reverse common primer: 5′-TACAACCCGCTTATCAATCCCA-3′;

[0064] CYP2D6 gene*5 locus:

[0065] Forward wild-type primer: 5'-GCACTTCAGCTTCTCGGTGC-3',

[0066] Forward mutant primer: 5'-AAAGGCCATCATCAGCTCCC-3',

[0067] Reverse common primer: 5′-ACATCTGCTCAGCCTCAACG-3′;

[0068] ABCB1 gene rs2032582 site:

[0069] Forward wild-type primer: 5'-ATTTAGTTTGACTCACCTTCCCAGA-3', forward mutant 1 primer: 5'-TCATATTTAGTTTGACTCACCTTCCCAGC-3', forward mutant 2 primer: 5'-TCAATCATATTTAGTTTGACTCACCTTCCCAGT-3', reverse common primer: 5'-ATCTATGGTTGGCAACTAACACTGTTAC-3'; ABCB1 gene rs1045642 site:

[0070] Forward wild-type primer: 5'-CCTCCTTTGCTGCCCTCACA-3',

[0071] Forward mutant primer: 5′-TTGGCCTCCTTTGCTGCCCTCACG-3′, reverse common primer: 5′-AGGCAAAGAAATAAAGCGACTG-3′;

[0072] CYP2D6 gene rs16947 site:

[0073] Forward wild-type primer: 5'-AGGTCAGCCACCACTATGCG-3',

[0074] Forward mutant primer: 5'-GAACAGGTCAGCCACCACTATGCA-3',

[0075] Reverse common primer: 5′-GGTTGGACCAGTGCATCACC-3′;

[0076] ABCB1 gene rs1128503 site:

[0077] Forward wild-type primer: 5'-CACTCTGCACCTTCAGGTTCAGA-3',

[0078] Forward mutant primer: 5′-TGCCCACTCTGCACCTTCAGGTTCAGG-3′, reverse common primer: 5′-GTTCACTTCAGTTACCCATCTCGA-3′;

[0079] ADRB2 gene rs1042718 site:

[0080] Forward wild-type primer: 5'-CCCATTCAGATGCACTGGTAGC-3',

[0081] Forward mutant primer: 5'-CTTGCCCATTCAGATGCACTGGTGCA-3',

[0082] Reverse common primer: 5′-CTGCTCCACCTGGCTAAGGTT-3′;

[0083] CREB1 gene rs2952768 site:

[0084] Forward wild-type primer: 5′-AGTGCTTTTTACTTTTATCTGAATGAT-3′, forward mutant primer: 5′-AAATAGTGCTTTTACTTTTATCTGAATGAC-3′, reverse common primer: 5′-CCAGGAGACAGGAGGGACAA-3′;

[0085] CYP2C19 gene rs4986893 site:

[0086] Forward wild-type primer: 5'-AAACTTGGCCTTACCTGGATC-3', forward mutant primer: 5'CAAAAAACTTGGCCTTACCTGGATT-3', reverse common primer: 5'-GTTGTTTCCAATCATTTAGCTTCA-3'; COMT gene rs4680 site:

[0087] Forward wild-type primer: 5′-GGATGGTGGATTTCGCTGGCG-3′, forward mutant primer: 5′-CAGCGGATGGTGGATTTCGCTGGCA-3′, reverse common primer: 5′-ACCTTGGCAGTTTACCCAGAGC-3′;

[0088] G6PD gene rs72554665 site:

[0089] Forward wild-type primer: 5′-GAAAATACGCCAGGCCTCAC-3′, forward mutant primer: 5′-GGGTGAAAATACGCCAGGCCTCAA-3′, reverse common primer: 5′-AAGCTCCCTGACGCCTATGA-3′;

[0090] CYP2D6 gene rs35742686 site:

[0091] Forward wild-type primer: 5'-GATGAGCTGCTAACTGAGCACAG-3', forward mutant primer: 5'-GCTGGATGAGCTGCTAACTGAGCACG-3', reverse common primer: 5'-AGAGCATACTCGGGACAGAACG-3'; FAAH gene rs3766246 site:

[0092] Forward wild-type primer: 5′-TCAAGACTTCTCCCCAAACACACT-3′, forward mutant primer: 5′-GGGTTCAAGACTTCTCCCCAAACACACC-3′, reverse common primer: 5′-TTTATAGAGGAGGGACTGTGCAAG-3′;

[0093] CYP2D6 gene rs28371725 site:

[0094] Forward wild-type primer: 5'-CCCCGCCTGTACCCTTC-3',

[0095] Forward mutant primer: 5′-TGGGCCCCCGCCTGTACCCTTT-3′, reverse common primer: 5′-GTTCTCTGCCGGGATGGTGAC-3′;

[0096] G6PD gene rs137852327 site:

[0097] Forward wild-type primer: 5′-GGCTTTCTCTCAGGTCAAGG-3′, forward mutant primer: 5′-CCTTGGCTTTCTCTCAGGTCAAGA-3′, reverse common primer: 5′-CACATAGAGGACGACGGCTG-3′;

[0098] CYP2C19 gene rs4244285 site:

[0099] Forward wild-type primer: 5′-TTCCCACTATCATTGATTATTTCCCG-3′, forward mutant primer: 5′-AATTTTCCCACTATCATTGATTATTTCCCA-3′, reverse common primer: 5′-AACTAGTCAATGAATCACAGATACGC-3′;

[0100] CYP2C9 gene rs1057910 site:

[0101] Forward wild-type primer: 5′-GCTGGTGGGGAGAAGGTCAAT-3′, forward mutant primer: 5′-GCAGGCTGGTGGGGAGAAGGTCAAG-3′, reverse common primer: 5′-AACCATCCTCTCTTTAAGTTTGC-3′;

[0102] CYP2D6 gene rs3892097 site:

[0103] Forward wild-type primer: 5'-GGGCGAAAGGGGCGTCC-3',

[0104] Forward mutant primer: 5′-GTTGGGGCGAAAGGGGCGTCT-3′, reverse common primer: 5′-TTCGCCAACCACTCCGGTG-3′;

[0105] G6PD gene rs398123546 site:

[0106] Forward wild-type primer: 5′-GAGCCAGATGCACTTCGTGC-3′, forward mutant primer: 5′-GCGGGAGCCAGATGCACTTCGTGT-3′, reverse common primer: 5′-GTCGCTGAGGGGACATGGTA-3′;

[0107] PTGS2 gene rs6500265 site:

[0108] Forward wild-type primer: 5′-GATACCCCACTGGGGAAAGGG-3′, forward mutant primer: 5′-GACTGATACCCCACTGGGGAAAGGA-3′, reverse common primer: 5′-TGGCTTAGGTATGTGCCTGTATG-3′;

[0109] FAAH gene rs324420 site:

[0110] Forward wild-type primer: 5′-ATAGAGCAGGCCCTGCCTTGG-3′, forward mutant primer: 5′-CGCCATAGAGCAGGCCCTGCCTTGT-3′, reverse common primer: 5′-ATTTCTTAAAAAGGCCAGCCTCCTT-3′;

[0111] CYP2E1 gene rs72559710 site:

[0112] Forward wild-type primer: 5′-CCGTGCATCACCACCATGC-3′, forward mutant primer: 5′-GTAGCCGTGCATCACCACCATGT-3′, reverse common primer: 5′-TCTAGAGCAACAGCAATACCCG-3′;

[0113] DRD2 gene rs1076560 site:

[0114] Forward wild-type primer: 5′-ACTGGCCCCTCCCTTTCC-3′, forward mutant primer: 5′-TCTCACTGGCCCCTCCCTTTCA-3′, reverse common primer: 5′-TGAGAGACAAGTCCCTGGTATTCA-3′;

[0115] G6PD gene rs137852331 site:

[0116] Forward wild-type primer: 5′-TCTCCACGATGATGCGGTT-3′, forward mutant primer: 5′-GGCTTCTCCACGATGATGCGGTC-3′, reverse common primer: 5′-GGGAGGGCGTCTGAATGAT-3′;

[0117] RYR1 gene rs111888148 site:

[0118] Forward wild-type primer: 5′-CACAGTTGCTACGATTGCCAC-3′, forward mutant primer: 5′-AGGGCACAGTTGCTACGATTGACAT-3′, reverse common primer: 5′-TATGAACTCCTAGGTAGGGGTCC-3′;

[0119] RYR1 gene rs193922832 site:

[0120] Forward wild-type primer: 5′-GCAGGTTCTCCACCATCTTCTC-3′, forward mutant primer: 5′-AGCCGCAGGTTCTCCACCATCTTCTT-3′, reverse common primer: 5′-TCCGATTCCAGAGCTGATGTT-3′;

[0121] OPRM1 gene rs1799971 site:

[0122] Forward wild-type primer: 5'-AACTTGTCCCACTTAGATGGCA-3', forward mutant primer: 5'-GGTCAACTTGTCCCACTTAGATGGCG-3', reverse common primer: 5'-CACGCACACGATGGAGTAGAG-3'; RYR1 gene rs193922816 site:

[0123] Forward wild-type primer: 5′-GAGCGGAGGATGGCGCG-3′, forward mutant primer: 5′-AAGGGAGCGGAGGATGGCGCA-3′, reverse common primer: 5′-GGCACAGGCAGAGGAACGAG-3′;

[0124] G6PD gene rs137852340 site:

[0125] Forward wild-type primer: 5'-GCCTTCCATCAGTCGGATACACA-3', forward mutant primer: 5'-CGATGCCTTCCATCAGTCGGATACACG-3', reverse common primer: 5'-GATGATCCTGGCGCACTAGCA-3'; RYR1 gene rs118192175 site:

[0126] Forward wild-type primer: 5′-GAGTGCCTCGGCCAGATCC-3′, forward mutant primer: 5′-GCTCGAGTGCCTCGGCCAGATCT-3′, reverse common primer: 5′-CCCAAGGGGATATGCAAAGTAA-3′;

[0127] CYB5R3 gene rs121965015 site:

[0128] Forward wild-type primer: 5′-ACCCTGATGACCACACTGTGTG-3′, forward mutant primer: 5′-AAGGACCCTGATGACCACACTGTGTA-3′, reverse common primer: 5′-CCCAGAATCTCAGCAGAGCTGT-3′;

[0129] G6PD gene rs137852314 site:

[0130] Forward wild-type primer: 5′-GATGATGCGGTTCCAGCC-3′, forward mutant primer: 5′-CCACGATGATGCGGTTCCAGCT-3′, reverse common primer: 5′-TCTGGGAACACAAGGCACG-3′;

[0131] CYP3A4 gene rs2242480 site:

[0132] Forward wild-type primer: 5′-TCCTCCCTCCTTCTCCATGTAC-3′, forward mutant primer: 5′-CACCTCCTCCCTCCTTCTCCATGTAT-3′, reverse common primer: 5′-CCCTTAGGGATTTGAGGGCTTCA-3′;

[0133] FAAH gene rs2295632 site:

[0134] Forward wild-type primer: 5′-CTGCTCTCAACAGGCTCCAAA-3′, forward mutant primer: 5′-AGCCCTGCTCTCAACAGGCTCCAAC-3′, reverse common primer: 5′-TCCCTGAGTCTGGACCTCCAT-3′;

[0135] RYR1 gene rs193922753 site:

[0136] Forward wild-type primer: 5'-GGTCTGAAGGAGAAAAGGTCCG-3', forward mutant primer: 5'-CAGAGGTCTGAAGGAGAAAAGGTCCT-3', reverse common primer: 5'-GAAATGATCAAGTCGGGCAAT-3'; RYR1 gene rs121918594 site:

[0137] Forward wild-type primer: 5′-ATCCGCGCCATCCTCCG-3′, forward mutant primer: 5′-GCGGATCCGCGCCATCCTCCA-3′, reverse common primer: 5′-CTCCTTCGCCAAGTTCCATC-3′;

[0138] UGT2B7 gene rs10028494 site:

[0139] Forward wild-type primer: 5'-GTTGTTTCTGTGCCTGGCCT-3', forward mutant primer: 5'-TGAAGTTGTTTCTGTGCCTGGCCG-3',

[0140] Reverse common primer: 5′-CAGGAAGATGAAAGGGTGGAGAT-3′;

[0141] CYP2D6 gene rs5030655 site:

[0142] Forward wild-type primer: 5'-GCAAGAAGTCGCTGGAGCAGT-3',

[0143] Forward mutant primer: 5'-CTGGGCAAGAAGTCGCTGGAGCAGG-3',

[0144] Reverse common primer: 5′-GTTGCTCACGGCTTTGTCCAAGAGA-3′;

[0145] PTGS2 gene rs9933632 site:

[0146] Forward wild-type primer: 5'-AGGCCATATGGTTTCCTTGAAAC-3',

[0147] Forward mutant primer: 5'-CAAAGGCCATATGGTTTCCTTGAAAA-3',

[0148] Reverse common primer: 5′-AGAGATGAGCTTATGGGGGCA-3′;

[0149] G6PD gene rs137852342 site:

[0150] Forward wild-type primer: 5'-CCACCTCTCATTCTCCACATAGAG-3',

[0151] Forward mutant primer: 5'-CATCCCACCTCTCATTCTCCACATAGAA-3',

[0152] Reverse common primer: 5'-ACACCCAAGGAGCCCATTC-3'.

[0153] In order to coordinate amplification efficiency, improve product peak shape, and facilitate capillary electrophoresis detection, we further made a series of specific changes or modifications to all primers.

[0154] For example, taking the rs1065852 site primer screening as an example, the site primers introduced different strengths of mismatches at the 5' end and 3' end of the wild-type primer and the mutant primer, respectively. The specific screening results are as follows: Figure 1 The three primers of rs1065852 locus were mixed in a ratio of 1:1:1, and the wild type, heterozygous and mutant samples determined by the gold standard were used as templates for amplification detection. The results showed correct results without any impurity peaks.

[0155] Taking the screening of primer combinations for some sites during the system construction process as an example, the optimization screening of the system primer combinations is described. Since there are certain differences between the primer combinations in the screening process of all sites and the final primer combinations (the differences include mismatches, positions, modifications, etc., and are not limited to the modifications), here, for example, the optimization of the first group of amplification systems is taken as an example to reflect the process of screening primer combinations, such as Figure 2 The statistical results are shown in the following table:

[0156]

[0157]

[0158] In summary, after screening the primers at each site individually and optimizing the combination of the primers at each site, it was determined that the four amplification systems finally adopted in this embodiment and the improved and optimized final primer sequences adopted are as follows:

[0159] CACNA1S gene rs772226819 site:

[0160] Forward wild-type primer:

[0161] Forward mutant primer:

[0162] Reverse common primer: 5'-ACCCAGAACTGGGGGACAAG-3' (SEQ ID NO.3)

[0163] CYP2D6 gene rs1065852 site:

[0164] Forward wild-type primer:

[0165] Forward mutant primer:

[0166] Reverse common primer: 5'-ACCCATTTGGTAGTGAGGCAGGT-3' (SEQ ID NO.6)

[0167] G6PD gene rs72554664 site:

[0168] Forward wild-type primer: 5'-GCAGCAGTGGGGTGAAAAT G C-3' (SEQ ID NO.7)

[0169] Forward mutant primer:

[0170] Reverse common primer: 5'-AGGGAGCCAGATGCACTTCGT-3' (SEQ ID NO.9)

[0171] BChE gene rs1803274 site:

[0172] Forward wild-type primer:

[0173] Forward mutant primer:

[0174] Reverse common primer: 5'-TCAGGCAAAGCGAGCTAATAACAA-3' (SEQ ID NO.12)

[0175] FAAH gene rs4141964 site:

[0176] Forward wild-type primer: 5'-ACCAAAGTAAGTCTGCACCTCC T GA-3' (SEQ ID NO. 13)

[0177] Forward mutant primer:

[0178] Reverse common primer: 5'-TACAACCCGCTTATCAATCCCA-3' (SEQ ID NO.15)

[0179] CYP2D6 gene*5 locus:

[0180] Forward wild-type primer: 5'-GCACTTCAGCTTCTCGGTGC-3' (SEQ ID NO.16)

[0181] Forward mutant primer: 5'-AAAGGCCATCATCAGCTCCC-3' (SEQ ID NO.17)

[0182] Reverse common primer: 5'-ACATCTGCTCAGCCTCAACG-3' (SEQ ID NO.18)

[0183] ABCB1 gene rs2032582 site:

[0184] Forward wild-type primer:

[0185] Forward mutant 1 primer:

[0186] Forward mutant 2 primer:

[0187] Reverse common primer: 5'-ATCTATGGTTGGCAACTAACACTGTTAC-3' (SEQ ID NO.22)

[0188] ABCB1 gene rs1045642 site:

[0189] Forward wild-type primer:

[0190] Forward mutant primer: Reverse common primer: 5'-AGGCAAAGAAATAAAGCGACTG-3' (SEQ ID NO.25)

[0191] CYP2D6 gene rs16947 site:

[0192] Forward wild-type primer: 5'-AGGTCAGCCACCACTATG A G-3' (SEQ ID NO.26)

[0193] Forward mutant primer:

[0194] Reverse common primer: 5'-GGTTGGACCAGTGCATCACC-3' (SEQ ID NO.28)

[0195] ABCB1 gene rs1128503 site:

[0196] Forward wild-type primer:

[0197] Forward mutant primer:

[0198] Reverse common primer: 5'-GTTCACTTCAGTTACCCATCTCGA-3' (SEQ ID NO.31)

[0199] ADRB2 gene rs1042718 site:

[0200] Forward wild-type primer:

[0201] Forward mutant primer:

[0202] Reverse common primer: 5'-CTGCTCCACCTGGCTAAGGTT-3' (SEQ ID NO.34)

[0203] CREB1 gene rs2952768 site:

[0204] Forward wild-type primer: 5'-AGTGCTTTTTACTTTTATCTGAA G GAT-3' (SEQ ID NO. 35)

[0205] Forward mutant primer:

[0206] Reverse common primer: 5'-CCAGGAGACAGGAGGGACAA-3' (SEQ ID NO.37)

[0207] CYP2C19 gene rs4986893 site:

[0208] Forward wild-type primer: 5'-AAACTTGGCCTTACCTGGA A C-3' (SEQ ID NO.38)

[0209] Forward mutant primer:

[0210] Reverse common primer: 5'-GTTGTTTCCAATCATTTAGCTTCA-3' (SEQ ID NO.40)

[0211] COMT gene rs4680 site:

[0212] Forward wild-type primer: 5'-GGATGGTGGATTTCGCTG TCG-3' (SEQ ID NO.41)

[0213] Forward mutant primer:

[0214] Reverse common primer: 5'-ACCTTGGCAGTTTACCCAGAGC-3' (SEQ ID NO.43)

[0215] G6PD gene rs72554665 site:

[0216] Forward wild-type primer: 5'-GAAAATACGCCAGGCCTC T C-3' (SEQ ID NO.44) forward mutant primer:

[0217] Reverse common primer: 5'-AAGCTCCCTGACGCCTATGA-3' (SEQ ID NO.46)

[0218] CYP2D6 gene rs35742686 site:

[0219] Forward wild-type primer: 5'-GATGAGCTGCTAACTGAGCA T AG-3' (SEQ ID NO.47)

[0220] Forward mutant primer: NO.48)

[0221] Reverse common primer: 5'-AGAGCATACTCGGGACAGAACG-3' (SEQ ID NO.49)

[0222] FAAH gene rs3766246 site:

[0223] Forward wild-type primer:

[0224] Forward mutant primer:

[0225] Reverse common primer: 5'-TTTATAGAGGAGGGACTGTGCAAG-3' (SEQ ID NO.52)

[0226] CYP2D6 gene rs28371725 site:

[0227] Forward wild-type primer:

[0228] Forward mutant primer: Reverse common primer: 5'-GTTCTCTGCCGGGATGGTGAC-3' (SEQ ID NO.55)

[0229] G6PD gene rs137852327 site:

[0230] Forward wild-type primer: 5'-GGCTTTCTCTCAGGTCA T GG-3' (SEQ ID NO.56)

[0231] Forward mutant primer:

[0232] Reverse common primer:

[0233] CYP2C19 gene rs4244285 site:

[0234] Forward wild-type primer:

[0235] Forward mutant primer:

[0236] Reverse common primer: 5'-AACTAGTCAATGAATCACAGATACGC-3' (SEQ ID NO.61)

[0237] CYP2C9 gene rs1057910 site:

[0238] Forward wild-type primer: 5'-GCTGGTGGGGAGAAGGTC G AT-3' (SEQ ID NO.62)

[0239] Forward mutant primer:

[0240] Reverse common primer: 5'-AACCATCCTCTCTTTAAGTTTGC-3' (SEQ ID NO.64)

[0241] CYP2D6 gene rs3892097 site:

[0242] Forward wild-type primer: 5'-GGGCGAAAGGGGCG A CC-3' (SEQ ID NO.65) forward mutant primer: Reverse common primer: 5'-TTCGCCAACCACTCCGGTG-3' (SEQ ID NO.67) G6PD gene rs398123546 site:

[0243] Forward wild-type primer: 5'-GAGCCAGATGCACTTCG A GC-3' (SEQ ID NO.68) forward mutant primer: Reverse common primer: 5'-GTCGCTGAGGGGACATGGTA-3' (SEQ ID NO.70) PTGS2 gene rs6500265 site:

[0244] Forward wild-type primer: 5'-GATACCCCACTGGGGAAAG T G-3' (SEQ ID NO.71) forward mutant primer:

[0245] Reverse common primer: 5'-TGGCTTAGGTATGTGCCTGTATG-3' (SEQ ID NO.73) FAAH gene rs324420 site:

[0246] Forward wild-type primer: 5'-ATAGAGCAGGCCCTGCCTT C G-3' (SEQ ID NO.74) forward mutant primer:

[0247] Reverse common primer: 5'-ATTTCTTAAAAAGGCCAGCCTCCTT-3' (SEQ ID NO.76) CYP2E1 gene rs72559710 site:

[0248] Forward wild-type primer: Forward mutant primer:

[0249] Reverse common primer: 5'-TCTAGAGCAACAGCAATACCCG-3' (SEQ ID NO.79) DRD2 gene rs1076560 site:

[0250] Forward wild-type primer: 5'-ACTGGCCCCTCCCTT A CC-3' (SEQ ID NO.80) forward mutant primer: Reverse common primer: 5'-TGAGAGACAAGTCCCTGGTATTCA-3' (SEQ ID NO.82) G6PD gene rs137852331 site:

[0251] Forward wild-type primer: Forward mutant primer: Reverse common primer: 5'-GGGAGGGCGTCTGAATGAT-3' (SEQ ID NO.85)

[0252] RYR1 gene rs111888148 site:

[0253] Forward wild-type primer:

[0254] Forward mutant primer:

[0255] Reverse common primer: 5'-TATGAACTCCTAGGTAGGGGTCC-3' (SEQ ID NO.88)

[0256] RYR1 gene rs193922832 site:

[0257] Forward wild-type primer: 5'-GCAGGTTCTCCACCATCT A CTC-3' (SEQ ID NO.89)

[0258] Forward mutant primer:

[0259] Reverse common primer: 5'-TCCGATTCCAGAGCTGATGTT-3' (SEQ ID NO.91)

[0260] OPRM1 gene rs1799971 site:

[0261] Forward wild-type primer: 5'-AACTTGTCCCACTTAGATG T CA-3' (SEQ ID NO.92)

[0262] Forward mutant primer:

[0263] Reverse common primer: 5'-CACGCACACGATGGAGTAGAG-3' (SEQ ID NO.94)

[0264] RYR1 gene rs193922816 site:

[0265] Forward wild-type primer: 5'-GAGCGGAGGATGGCG A G-3' (SEQ ID NO.95)

[0266] Forward mutant primer:

[0267] Reverse common primer: 5'-GGCACAGGCAGAGGAACGAG-3' (SEQ ID NO.97)

[0268] G6PD gene rs137852340 site:

[0269] Forward wild-type primer: 5'-GCCTTCCATCAGTCGGATAC T CA-3' (SEQ ID NO.98)

[0270] Forward mutant primer:

[0271] Reverse common primer: 5'-GATGATCCTGGCGCACTAGCA-3' (SEQ ID NO.100)

[0272] RYR1 gene rs118192175 site:

[0273] Forward wild-type primer: 5'-GAGTGCCTCGGCCAGA A CC-3' (SEQ ID NO.101)

[0274] Forward mutant primer:

[0275] Reverse common primer: 5'-CCCAAGGGGATATGCAAAGTAA-3' (SEQ ID NO.103)

[0276] CYB5R3 gene rs121965015 site:

[0277] Forward wild-type primer: 5'-ACCCTGATGACCACACTGT T TG-3' (SEQ ID NO. 104)

[0278] Forward mutant primer:

[0279] Reverse common primer: 5'-CCCAGAATCTCAGCAGAGCTGT-3' (SEQ ID NO.106)

[0280] G6PD gene rs137852314 site:

[0281] Forward wild-type primer: 5'-GATGATGCGGTTCCAG A C-3' (SEQ ID NO.107)

[0282] Forward mutant primer:

[0283] Reverse common primer: 5'-TCTGGGAACACAAGGCACG-3' (SEQ ID NO.109)

[0284] CYP3A4 gene rs2242480 site:

[0285] Forward wild-type primer:

[0286] Forward mutant primer:

[0287] Reverse common primer: 5'-CCCTTAGGGATTTGAGGGCTTCA-3' (SEQ ID NO.112)

[0288] FAAH gene rs2295632 site:

[0289] Forward wild-type primer:

[0290] Forward mutant primer:

[0291] Reverse common primer: 5'-TCCCTGAGTCTGGACCTCCAT-3' (SEQ ID NO.115)

[0292] RYR1 gene rs193922753 site:

[0293] Forward wild-type primer: 5'-GGTCTGAAGGAGAAAAGGTC A G-3' (SEQ ID NO.116)

[0294] Forward mutant primer:

[0295] Reverse common primer: 5'-GAAATGATCAAGTCGGGCAAT-3' (SEQ ID NO.118)

[0296] RYR1 gene rs121918594 site:

[0297] Forward wild-type primer: 5'-ATCCGCGCCATCCTC T G-3' (SEQ ID NO.119)

[0298] Forward mutant primer:

[0299] Reverse common primer: 5'-CTCCTTCGCCAAGTTCCATC-3' (SEQ ID NO.121)

[0300] UGT2B7 gene rs10028494 site:

[0301] Forward wild-type primer: 5'-GTTGTTTCTGTGCCTGG T CT-3' (SEQ ID NO. 122)

[0302] Forward mutant primer:

[0303] Reverse common primer: 5'-CAGGAAGATGAAAGGGTGGAGAT-3' (SEQ ID NO.124)

[0304] CYP2D6 gene rs5030655 site:

[0305] Forward wild-type primer: 5'-GCAAGAAGTCGCTGGAG G AGT-3' (SEQ ID NO.125)

[0306] Forward mutant primer:

[0307] Reverse common primer: 5'-GTTGCTCACGGCTTTGTCCAAGAGA-3' (SEQ ID NO.127)

[0308] PTGS2 gene rs9933632 site:

[0309] Forward wild-type primer: 5'-AGGCCATATGGTTTCCTTG T AAC-3' (SEQ ID NO. 128)

[0310] Forward mutant primer:

[0311] Reverse common primer: 5'-AGAGATGAGCTTATGGGGGCA-3' (SEQ ID NO.130)

[0312] G6PD gene rs137852342 site:

[0313] Forward wild-type primer:

[0314] Forward mutant primer:

[0315] Reverse common primer: 5'-ACACCCAAGGAGCCCATTC-3' (SEQ ID NO.133)

[0316] Among them, 1. The single underline “-” represents that each primer has 1 to 3 bases changed at the -2 to -5 position of the 3' end of the primer.

[0317] 2. The double underline "=" indicates that the sequence of each primer after position -15 at the 3' end of the primer has been modified, including adding other sequences at the end and changing part of the base sequence.

[0318] 3. All common primers for detection sites are fluorescently labeled with FAM or HEX at the 5' end. The specific labeling conditions are shown in the following table:

[0319]

[0320]

[0321] Example 3

[0322] The present invention is used to detect gene polymorphisms related to anesthetic medication in clinical samples.

[0323] In this example, the system of the present invention is used to prepare a kit to directly perform amplification detection on a blood sample, specifically a sample of a patient with knee replacement (patient, female, 68 years old, Department of Orthopedics, The First Affiliated Hospital of Zhengzhou University) is used as an example for amplification detection. At the same time, the amplification detection results of the kit are verified by sequencing, thereby illustrating the effectiveness, specificity and accuracy of the primer system and kit of the present invention.

[0324] 1. Detection system

[0325] The kit of the present invention comprises PCR Master Mix, quality control products, and internal standards. The main components of PCR Master Mix include hot start DNA Taq enzyme, amplification buffer, and amplification primers for each site.

[0326] 2. Detection Method

[0327] (1) Preparation of PCR amplification system

[0328] The system includes the primer combinations of sequences determined in Example 2 as shown in SEQ ID NO. 1-133, which were respectively carried out at Sangon Biotech (Shanghai) Co., Ltd., and all primers were mixed in equal proportions according to experimental exploration to prepare 15× primer mix.

[0329] The system also includes enzymes and PCR amplification buffer, wherein the PCR amplification buffer contains dATP, dTTP, dCTP, dGTP, Mg 2+ wait.

[0330] Prepare the PCR amplification system according to the components in the table below. After oscillation and mixing, divide the system into smaller portions according to the number of samples, with 14.5 μL per tube.

[0331] Component name Amount of each component per 15μL system (μL) PCR Amplification Buffer 7.5 15× Primer mix1 / 2 / 3 / 4 1 Enzymes 0.2 template 0.5 water 5.8 total 15

[0332] (2) Add template

[0333] Add 0.5 μL of the blood sample to be tested to the corresponding PCR reaction tube. At the same time, set up a quality control (quality control: 1 μL quality control product, negative control: 1 μL nucleic acid-free pure water).

[0334] (3) PCR amplification

[0335] Place each reaction tube into the reaction tank of the PCR amplification instrument and set the reaction system to 15 μL.

[0336] PCR amplification was performed according to the following reaction procedure:

[0337]

[0338] (4) Capillary electrophoresis detection of amplified products

[0339] Prepare a sample mixture containing molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) × number of samples to be tested, vortex and mix for 10-15 seconds; use a pipette to dispense 9 μL of formamide and internal standard mixture into each test well; take 1 μL of amplification product and add it to the formamide and internal standard mixture, and cover the plate with a rubber cover. Perform the test according to the steps in the genetic analyzer user manual.

[0340] (5) Data analysis

[0341] Import relevant files into GeneMapper software, input the raw data (.fsa file) from the detector, and analyze the data.

[0342] 3. Sanger sequencing

[0343] According to the final SNP site information, the gene sequence of each site is searched, and the sequencing primers are designed in the upstream and downstream regions of the corresponding SNP site at least 100bp (synthesized by Sangon Biotech (Shanghai) Co., Ltd.). The designed sequencing primers for each site are used to amplify the fragments of each site of the target sample. After electrophoresis detection to determine whether there is an amplified product and the size of the target fragment is correct, it is sent to Sangon Biotech (Shanghai) Co., Ltd. for Sanger sequencing.

[0344] 4. Comparison of CE results and sequencing results

[0345] According to the capillary electrophoresis detection chart, the results of the amplification test of the clinical samples by the kit are analyzed and the data obtained by sequencing by the sequencing company are analyzed to obtain the results of sequencing at each site of the clinical samples.

[0346] 5. Results

[0347] The test kit of the present invention is used to detect the typing results of 44 polymorphic sites in clinical blood samples ( Figure 3 ) and the sequencing typing results are shown in the following table:

[0348]

[0349] The above test results show that the results of the genotyping test of clinical blood samples by the kit provided by the present invention are consistent with the results of amplification sequencing typing. Therefore, the test results of the kit are reliable compared with the gold standard. The present invention can be used to perform multiple amplification detection on 44 SNP sites corresponding to 24 drugs in 7 categories commonly used in the Department of Anesthesiology, and obtain the genotyping of patients for related anesthetic drugs. The obtained genotyping results are used to realize the evaluation of adverse reactions and toxicity warnings, efficacy evaluation, etc. of anesthetic drugs by patients, provide more genetic reference information for clinicians, and provide effective evaluation indicators for the individualized use of anesthetic drugs.

[0350] Example 4

[0351] The present invention was used to detect gene polymorphisms related to anesthetic medication in 204 blood samples.

[0352] This example uses the system of the present invention to prepare a kit, and directly performs amplification detection on 204 blood samples. At the same time, the amplification detection results of the kit are verified by sequencing, further illustrating the effectiveness, specificity and accuracy of the primer system and kit of the present invention. The specific operation process is the same as that of Example 3.

[0353] The test results showed that the results of the 204 samples tested by the present invention were completely consistent with the sequencing results, with an accuracy rate of 100%.

[0354] The test results except *5, the genotypes and frequencies of the remaining 43 SNP sites were statistically analyzed, and the Hardy-Weinberg equilibrium was calculated. All sites conformed to the Hardy-Weinberg equilibrium. The results are shown in the following table:

[0355]

[0356]

[0357]

[0358]

[0359] The above embodiments are only used to illustrate the technical solutions of the present invention, rather than to limit the same. Although the present invention has been described in detail with reference to the aforementioned embodiments, those skilled in the art should understand that the technical solutions described in the aforementioned embodiments may still be modified, or some of the technical features may be replaced by equivalents. However, these modifications or replacements do not deviate the essence of the corresponding technical solutions from the principles and scope of the technical solutions of the embodiments of the present invention.

Claims

1. A primer composition for detecting gene polymorphism related to anesthetic medication, characterized in that The primer composition comprises 133 primers, and the sequences thereof are shown as SEQ ID NO.1-133.

2. The primer composition according to claim 1, characterized in that The primers in the primer composition further include one of fluorescent group modification, phosphorylation modification, thiophosphorylation modification, locked nucleic acid modification or peptide nucleic acid modification.

3. A composite amplification system for detecting gene polymorphisms related to anesthetic drugs, characterized in that The composite amplification system comprises a primer combination shown in SEQ ID NO. 1-133, and the composite amplification system is a system combining multiple fluorescent ARMS PCR with capillary electrophoresis detection.

4. A kit for detecting gene polymorphisms related to anesthetic medication, characterized in that The kit comprises a primer combination or a composite amplification system shown in SEQ ID NO. 1-133.

5. A method for detecting gene polymorphism related to anesthetic medication, characterized in that The method is as follows: the sample to be tested is subjected to PCR amplification and capillary electrophoresis detection using the sequence shown in SEQ ID NO.1-133 or the composite amplification system or the above-mentioned kit, and then the presence or absence of a specific genotype at a specific site of the sample is determined by the presence or absence of a specific fluorescent marker and amplification product of a specific length.

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