Multiplex amplification system, detection method and kit for genetic typing of anesthetic drugs

Abstract

The application provides a composite amplification system, a kit and a detection method for anesthetic drug-related gene polymorphism detection, and relates to the technical field of drug gene detection.The application designs a series of specific primer compositions and a composite amplification system shown in sequences SEQ ID NO.1-133 for 44 SNP sites corresponding to 24 drugs in 7 categories;the kit obtained by using the primer composition or the composite amplification system has very high sensitivity for the 44 SNP sites related to anesthetic drugs, the detection result is accurate, and the kit can be consistent with the gold standard;at the same time, the kit and the detection method provided by the application support blood or blood card direct filling, do not need to pretreat the sample to be detected, can directly obtain the detection result, the operation steps are simple, the time consumption is short, can greatly reduce the cost, has the advantages of high sensitivity, high resolution, high throughput and the like.Using the application, the genotyping of the patient to the related anesthetic drug can be quickly and accurately obtained, the adverse reaction and toxicity warning, the efficacy evaluation and other evaluations of the patient to the anesthetic drug are realized, more gene reference information of the patient is provided for the clinician, and effective evaluation indexes are provided for the individual use of the anesthetic drug.

Description

TECHNICAL FIELD

[0001] The present application relates to the technical field of drug gene detection, and particularly relates to a composite amplification system for anesthetic drug genotyping, a detection method and a kit. BACKGROUND

[0002] Modern anesthesiology began in 1846. In the clinical application of anesthetic drugs, when different patients use the same kind and same dose of anesthetic drugs, there are obvious individual differences in the time of consciousness disappearance, the time of recovery, the depth of anesthesia and the incidence of adverse reactions. Many studies have shown that genetic factors are an important reason for the differences in pharmacokinetics and pharmacodynamics reactions among individuals. Pharmacogenomics is a discipline that studies the polymorphism of genes related to drug metabolism and pharmacodynamics and individual differences in drug reactions, aiming to find drug metabolism enzymes, drug transport proteins and action targets related to individual differences in drug reactions. Pharmacogenomic research of anesthetic drugs can provide an important basis for individualized use of anesthetic drugs and prevention of adverse reactions, and improve the safety of anesthetic drugs in the surgical process.

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

[0004] Although these technologies can effectively detect single nucleotide polymorphisms (SNPs), there are still some limitations in practical application. For example, the operation process of some methods is complex and requires restriction enzyme digestion; some require two-step PCR amplification reaction. In addition, although some new technologies have the advantages of high throughput and easy automation, they usually require expensive instruments and equipment. Some technologies have shortcomings in repeatability, result interpretation, number of detection sites, throughput, detection period and cost, such as poor repeatability, difficult result interpretation, few detection sites, low detection throughput, long detection period and high detection cost. SUMMARY

[0005] (I) Technical problems solved

[0006] In view of the deficiencies of the prior art, the present application provides a composite amplification system, kit and detection method for anesthetic drug-related gene polymorphism detection, which has high throughput, strong specificity, high sensitivity, low cost and simple operation.

[0007] (II) Technical solutions

[0008] To achieve the above object, the present application is implemented by the following technical solutions:

[0009] In a first aspect, the present application provides a primer composition for anesthetic drug related gene polymorphism detection, which comprises 133 primers, and the sequences of the primers are shown in SEQ ID NO. 1-133.

[0010] The above primer group is obtained by a large number of screening and analysis, primer design for 44 SNP sites corresponding to 24 drugs of 7 categories commonly used in the anesthesiology department, and the SNP sites are as follows:

[0011]

[0012] Further, the primers in the primer group further comprise one of a fluorescent group modification, a phosphorylation modification, a thiophosphorylation modification, a locked nucleic acid modification or a peptide nucleic acid modification.

[0013] In a second aspect, the present application provides a composite amplification system for anesthetic drug related gene polymorphism detection, which comprises the primer composition shown in SEQ ID NO. 1-133, and the composite amplification system is a system of multiplex fluorescence ARMS PCR combined with capillary electrophoresis detection.

[0014] Further, in the system, the detection adopts capillary electrophoresis detection, and the amplification adopts multiplex allele-specific PCR amplification.

[0015] In a third aspect, the present application further provides a kit for anesthetic drug related gene polymorphism detection, which comprises the primer composition shown in SEQ ID NO. 1-133 or the composite amplification system.

[0016] Further, the detection kit adopts the method of allele-specific PCR (ASPCR) combined with quantitative fluorescent PCR (QF-PCR) to amplify the target site, detects the amplification product by capillary electrophoresis, and completes the detection of the genotyping of the target site; for each detection site, three primers are set, and one specific primer of different lengths and one fluorescent-labeled downstream primer are set for two types; each specific primer can only combine with the DNA template of the corresponding genotype and be amplified; after completing the PCR amplification and capillary electrophoresis detection, whether the specific site of the sample has the specific genotype can be determined by the specific fluorescent label and the presence or absence of the specific length of the amplification product; in general, the target fragment amplified by the wild type primer is smaller than the fragment amplified by the mutant primer by 4 bp.

[0017] Further, the kit further comprises a hot-start DNA Taq enzyme, 2x PCR amplification buffer.

[0018] Further, the kit further comprises a quality control product and related reagents required for capillary detection.

[0019] In a fourth aspect, the present application provides a detection method for anesthetic drug-related genetic polymorphism, which comprises: using the primer composition or the composite amplification system or the kit for PCR amplification and capillary electrophoresis detection of the sample to be tested, and then determining whether a specific genotype exists at a specific site of the sample by specific fluorescence labeling and the presence or absence of a specific length of amplification product.

[0020] (III) Advantages

[0021] The present application provides a composite amplification system, a kit and a detection method for anesthetic drug-related genetic polymorphism, which has the following advantages compared with the prior art:

[0022] 1. According to the principle of allele-specific PCR, the present application first designs specific primer sequences for 44 SNP sites corresponding to 24 drugs of 7 categories, and further makes a series of specific modifications to all primers to coordinate amplification efficiency, improve product peak type, and improve detection efficiency, etc. Finally, the primer composition comprising the sequences of SEQ ID NO. 1-133 is obtained; the primer composition covers a wide range of sites and can realize multiple specific detection of multiple SNP sites.

[0023] 2. The kit provided by the present application has high accuracy of detection results 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 application supports direct filling of blood or blood cards without pretreatment of the sample to be tested, and can directly obtain the detection results, and the operation steps are simple.

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

[0025] 4. The composite amplification system, the kit and the detection method for anesthetic drug-related genetic polymorphism provided by the present application can perform multiple amplification detection on 44 SNP sites corresponding to 24 drugs of 7 categories commonly used in the anesthesiology department, realize early warning of toxicity, malignant symptoms, efficacy evaluation, etc. of anesthetic drugs for patients, and provide more genetic information of patients for clinicians and effective evaluation indexes for individual use of anesthetic drugs. BRIEF DESCRIPTION OF DRAWINGS

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

[0027] Figure 2 First group of amplification system construction primer combination optimization (from top to bottom are combination 1, combination 2, combination 3 respectively).

[0028] Figure 3 The results of capillary electrophoresis detection of the kit amplification of the clinical sample of example 3. DETAILED DESCRIPTION

[0029] To make the purposes, technical solutions and advantages of the embodiments of the present application clearer, the technical solutions in the embodiments of the present application will be described clearly and completely below in combination with the embodiments of the present application. Obviously, the described embodiments are part of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those of ordinary skill in the art without creative labor fall within the scope of protection of the present application.

[0030] Example 1

[0031] Screening and determination of anesthetic drug-related gene sites

[0032] In this embodiment, the screening of the effect target of the antidepressant drug efficacy was performed according to the Drug Metabolism Enzyme and Drug Action Target Gene Detection Technology Guidelines (Trial) expert guidelines formulated by the National Health and Family Planning Commission Individualized Medicine Detection Technology Expert Committee, CPIC, diagnosis and treatment guidelines, FDA drug instructions and the clinical evidence level in the pharmGKB database.

[0033] In this embodiment, 24 anesthetic-related drugs of 7 categories were screened. The drug classification and the drug action target include the following table:

[0034]

[0035]

[0036]

[0037] Further, according to the characteristics of the sequences of the sites, the size arrangement of the sites in the system and the difficulty of the system optimization, the action targets of 44 SNP sites were finally selected for subsequent detection, and the genes and information are as follows:

[0038]

[0039]

[0040] Example 2

[0041] Construction and optimization of detection system for anesthetic drug related gene polymorphism

[0042] (1) Primers for initial design of selection sites

[0043] According to the principle of allele-specific PCR, each specific primer can only bind to the DNA template of the corresponding genotype and be amplified. The primer sequences are 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 sites:

[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 primer 1: 5'-TCATATTTAGTTTGACTCACCTTCCCAGC-3', forward mutant primer 2: 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'; rs4680 site of COMT gene:

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

[0088] rs72554665 site of G6PD gene:

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

[0090] rs35742686 site of CYP2D6 gene:

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

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

[0093] rs28371725 site of CYP2D6 gene:

[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 rs13785233l 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 rs1219650 site:

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

[0129] G6PD gene rs13785231 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 rs19392275 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] To coordinate the amplification efficiency, improve the product peak type, and facilitate capillary electrophoresis detection, we further made a series of specific modifications or modifications to all primers.

[0154] For example, the rs1065852 site primer screening, the 5' and 3' ends of the wild type primer and the mutant primer were introduced with different intensity mismatches, and the screening results were as follows Figure 1 The three primers of rs1065852 site were mixed in a ratio of 1:1:1, and the wild, heterozygous, and mutant samples determined by the gold standard were used as templates for amplification detection. The results showed that it was correct and there was no impurity peak.

[0155] For example, the screening of part of the site primer combination in the system construction process illustrates the optimization and screening of the system primer combination. Due to the differences between the primer combination and the final primer combination during the screening process of all sites (the differences include mismatches, positions, modifications, etc., and are not limited to the modifications described), here, for example, the first group of amplification system optimization is used to reflect the process of screening primer combination, as follows Figure 2 The results are shown in the following table:

[0156]

[0157]

[0158] In summary, after the individual screening of each site primer and the optimization of each site primer combination, the final four amplification systems were determined in this embodiment, and the final primer sequences used after improvement and optimization 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 site:

[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 locus:

[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 locus:

[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 locus:

[0223] Forward wild-type primer:

[0224] Forward mutant primer:

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

[0226] CYP2D6 gene rs28371725 locus:

[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 rs3981235 position 546:

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

[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 position:

[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 position:

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

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

[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 locus:

[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 locus:

[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 locus:

[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 locus:

[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] 1. The "-" single underline represents that each primer is changed by 1 to 3 bases at the 3' end -2 to -5 of the primer.

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

[0318] 3. All detection site common primers are labeled with FAM or HEX fluorescence at the 5' end, and the specific labeling conditions are shown in the following table:

[0319]

[0320]

[0321] Example 3

[0322] The clinical sample is detected for anesthetic drug related gene polymorphism by using the application.

[0323] This example uses the kit prepared by the system of the application to directly amplify and detect the blood sample. Specifically, a knee replacement patient sample (patient, female, 68 years old, Department of Orthopedics, the First Affiliated Hospital of Zhengzhou University) is taken as an example for amplification and detection. At the same time, the amplification and detection results of the kit are verified by sequencing method, thereby illustrating the effectiveness, specificity and accuracy of the primer system and kit of the application.

[0324] 1. Detection system

[0325] The kit of the application comprises PCR Master Mix, quality control, and internal standard. The main components of the PCR Master Mix include hot-start DNA Taq enzyme, amplification buffer, and each site amplification primer, etc.

[0326] 2. Detection method

[0327] (1)PCR amplification system preparation

[0328] The system contains primer composition with sequences determined in Example 2 as shown in SEQ ID NO. 1-133, respectively, prepared in Shenguo Bioengineering (Shanghai) Co., Ltd., and all primers are mixed in equal proportions according to experimental exploration to prepare 15x primer mix.

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

[0330] Prepare the PCR amplification system according to the following table components, shake and mix, according to the sample quantity, dispense 14.5 μL per tube.

[0331] Component name Amount of each component used per 15 μL system (μL) PCR amplification buffer 7.5 15 x primer mix 1 / 2 / 3 / 4 1 Enzyme 0.2 Template 0.5 Water 5.8 Total 15

[0332] (2) Add template

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

[0334] (3) PCR amplification

[0335] Put each reaction tube into the PCR amplification instrument reaction slot, and set the reaction system to 15 μL.

[0336] Perform PCR amplification according to the following reaction program:

[0337]

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

[0339] Prepare a loading mixture mixed with molecular weight internal standard and formamide: (0.5 μL molecular weight internal standard + 8.5 μL formamide) x number of detection samples, vortex and mix for 10-15 seconds; use a pipette to dispense 9 μL of formamide and internal standard mixture into each detection hole; take 1 μL of amplification product and add it to the formamide and internal standard mixture, cover with a sealing plate cover. Perform detection according to the steps in the Genetic Analyzer User Manual.

[0340] (5) Data analysis

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

[0342] 3. Sanger sequencing

[0343] According to the SNP site information finally determined, the gene sequence of each site was searched, and the sequencing primer was designed in the region of at least 100 bp or more upstream and downstream of the corresponding SNP site (synthesized by Sheng Wu Bioengineering (Shanghai) Co., Ltd.). The designed sequencing primer of each site was used to amplify the fragment of each site of the target sample, and after electrophoresis detection, whether the amplification product and the size of the target fragment were correct were determined, and then Sheng Wu Bioengineering (Shanghai) Co., Ltd. was sent to perform Sanger sequencing.

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

[0345] According to the capillary electrophoresis detection chart, the typing results of the clinical sample amplified and detected by the kit were analyzed. The data obtained by sequencing the company were analyzed, and the typing results of the sequencing of each site of the clinical sample were obtained.

[0346] 5. Results

[0347] The typing results of the 44 polymorphic sites of the clinical blood sample detected by the kit of the present application ( Figure 3 ) and the sequencing typing results are shown in the following table:

[0348]

[0349] The above detection results show that the results of the genotyping detection of the clinical blood sample by the kit of the present application are consistent with the results of the amplification sequencing typing. Therefore, the detection results of the kit are reliable compared with the gold standard. The present application can be used to perform multiplex amplification detection on 44 SNP sites corresponding to 24 drugs of 7 categories commonly used in the department of anesthesiology, to obtain the genotyping of the patient to the related anesthetic drugs, and to realize the adverse reaction and toxicity warning, efficacy evaluation and other evaluations of the patient to the anesthetic drugs by using the obtained genotyping results, to provide more genetic reference information for the clinician, and to provide effective evaluation index for the individual use of anesthetic drugs.

[0350] Example 4

[0351] 204 blood samples were detected for anesthetic drug related gene polymorphism by the present application.

[0352] In this embodiment, the kit was prepared by using the system of the present application, and 204 blood samples were directly amplified and detected, and at the same time, the amplification and detection results of the kit were verified by using the sequencing method, to further illustrate the effectiveness, specificity and accuracy of the primer system and the kit of the present application. The specific operation process is the same as that of example 3.

[0353] The detection results show that the results of the 204 samples detected by the present application are completely consistent with the sequencing results, and the accuracy is 100%.

[0354] The genotypes and frequencies of the remaining 43 SNP sites except for 5 were statistically detected, and the Hardy-Weinberg balance was calculated. All sites met the Hardy-Weinberg balance, and the results are shown in the following table:

[0355]

[0356]

[0357]

[0358]

[0359] The above examples are only used to illustrate the technical solutions of the present application, but not to limit it; although the present application has been described in detail with reference to the foregoing examples, those skilled in the art should understand that the technical solutions recorded in the foregoing examples can still be modified, or some technical features can be replaced by equivalents; and these modifications or replacements do not make the essence of the corresponding technical solutions deviate from the principles and scope of the technical solutions of the embodiments of the present application.

Claims

1. A primer composition for detecting polymorphisms in genes related to anesthetic drugs, characterized in that: The sequence of the primer composition is shown in SEQ ID NO.1-133.

2. The primer composition according to claim 1, characterized in that, The primers in the primer composition may also contain one of the following modifications: fluorescent group modification, phosphorylation modification, thiophosphorylation modification, locked nucleic acid modification, or peptide nucleic acid modification.

3. A multiplex amplification system for detecting polymorphisms in anesthetic drug-related genes, characterized in that, The multiplex amplification system contains the primer composition shown in SEQ ID NO.1-133, and the multiplex amplification system is a system for multiplex fluorescent ARMS PCR combined with capillary electrophoresis detection.

4. A kit for detecting gene polymorphisms related to anesthetic drugs, characterized in that, The kit contains the primer composition shown in SEQ ID NO.1-133 or the multiplex amplification system of claim 3, and the kit detects untreated whole blood or blood card samples.

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