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CN-121992089-A - CYP2C19 genotyping multiplex PCR screening method

CN121992089ACN 121992089 ACN121992089 ACN 121992089ACN-121992089-A

Abstract

The invention discloses a CYP2C19 genotyping multiplex PCR screening method which comprises the following steps of extracting target DNA samples from blood by a cleavage method, carrying out PCR amplification, mixing the DNA samples, primers, nuclease, reaction buffer solution, dNTPs, fluorescent dye and fluorescent label probes, carrying out PCR amplification, carrying out electrophoresis detection, placing a PCR reaction solution after S2 detection in a reaction tube, then placing the reaction tube in a fluorescent quantitative PCR instrument, collecting fluorescent signals at 75 ℃, and judging the result. The detection prevention of the scheme is adopted for detection, the stability of the obtained detection result is high, and the drug substitution condition corresponding to CYP2C19 genotypes in different human bodies can be rapidly and accurately judged so as to facilitate subsequent treatment. Through the setting of the PCR amplification parameters of the scheme, the detection accuracy can be improved, meanwhile, the amplification result test is carried out after the amplification, the products entering the tester are ensured to be amplified successfully, and the experimental efficiency and accuracy are improved.

Inventors

  • WANG HUAIXIN
  • LIU BING
  • CHEN WEILI

Assignees

  • 合肥安为康医学检验有限公司

Dates

Publication Date
20260508
Application Date
20251219

Claims (7)

  1. 1. A method for screening CYP2C19 genotyping multiplex PCR, which is characterized by comprising the following steps: S1, DNA extraction, namely extracting a target DNA sample from blood by a cleavage method; S2, PCR amplification, namely mixing the DNA sample, the primer, the nuclease, the reaction buffer solution, dNTPs, fluorescent dye and fluorescent marked probe, carrying out PCR amplification, and carrying out electrophoresis detection; s3, placing the PCR reaction solution detected by the S2 into a reaction tube, then placing the reaction tube into a fluorescent quantitative PCR instrument, and collecting fluorescent signals at 75 ℃; S4, judging the result, namely recording the Ct value (marked as CtF) of each detection FAM channel and the Ct value (marked as CtV) of the VIC channel, and calculating the absolute value delta Ct= |CtF-CtV |; If ΔCt <2.5, the locus is judged as heterozygous mutant; If DeltaCt >2.5, it is judged that the locus is judged to be homozygous wild type when CtF < CtV, and homozygous mutant when CtV < CtF; And determining the gene polymorphism condition of the CYP2C19 to be detected through specific values of delta Ct, ctV and CtF, thereby determining whether the reaction of the population carrying the detected gene to the drug is slow metabolism type, fast metabolism type or intermediate metabolism type.
  2. 2. The method for screening CYP2C19 genotyping multiplex PCR according to claim 1, wherein PCR amplification conditions comprise pre-denaturation at 90-95 ℃ for 15-18min, denaturation at 92-94 ℃ for 30-35s, annealing at 62-66 ℃ for 30-35s, elongation at 70-75 ℃ for 1-3min, total of 35-40 cycles, and elongation at 70-75 ℃ for 5-8min, and taking The products were subjected to 3% agarose gel electrophoresis.
  3. 3. The method for screening for multiple PCR of CYP2C19 genotyping according to claim 1, wherein said fluorescent dye is selected from one or more of Evagreen, sybrgreen, LC Green Plus.
  4. 4. The method for screening for multiple PCR of CYP2C19 genotyping according to claim 1, wherein said fluorescent label probe is at least one of TAMRA, FAM, HEX, NED, ROX, texas Red, cy5, cy 7.
  5. 5. The method for screening for multiple PCR of CYP2C19 genotyping according to claim 1, wherein the sequence of the fluorescent label probe is matched with the wild type sequence of the gene to be tested.
  6. 6. The method for screening for CYP2C19 genotyping multiplex PCR according to claim 1, wherein the primer comprises an upstream primer and a downstream primer.
  7. 7. The method for screening multiple CYP2C19 genotyping PCR according to claim 1, wherein said fluorescent quantitative PCR instrument is one of LightCycler 480, qiagen router-Gene Q, io-Rad CFX Maestr.

Description

CYP2C19 genotyping multiplex PCR screening method Technical Field The invention relates to the technical field of CYP2C19 genotyping screening, in particular to a CYP2C19 genotyping multiplex PCR screening method. Background CYP2C19 genotyping refers to the determination of the type of an individual's ability to metabolize certain drugs by analyzing specific sites of the CYP2C19 gene. By knowing the patient's CYP2C19 genotype, drug dosage adjustments can be optimized, thereby improving drug efficacy and reducing the risk of adverse reactions. Enzymes encoded by the CYP2C19 gene are involved in the metabolism of many drugs in the liver, including clozapine, clonazepam, chlorpromazine, chlorzoxazone, tramadol, and the like. Depending on the CYP2C19 genotype, individuals can be classified into the following drug metabolism types: normal metabolizers these individuals carry two normal functioning CYP2C19 alleles with normal metabolic rates for most drugs. These individuals are typically caused by a significant decrease or complete loss of CYP2C19 enzyme activity due to the nonfunctional or absent CYP2C19 alleles. These individuals may have a slower metabolic rate when using some drugs and may need to reduce the dosage or consider other treatment options. These individuals often carry multiple functional replications of the CYP2C19 allele, leading to increased CYP2C19 enzymatic activity. This may lead to rapid metabolism of some drugs, requiring higher doses to achieve effective therapeutic levels. Intermediary metabolizers, these individuals carry a normal functioning CYP2C19 allele and a nonfunctional or reduced function allele. Their drug metabolism rate is intermediate between those of normal and slow metabolism. The CYP2C19 genotype can be determined by molecular biological techniques such as PCR and sequencing. The genotype information is very important for personalized medicine treatment, and can help doctors optimize medicine treatment schemes, avoid side effects and improve treatment effects. It is therefore necessary to establish a PCR screening method to detect the CYP2C19 genotype in order to determine the metabolic status of the patient. Disclosure of Invention The invention aims to provide a CYP2C19 genotyping multiplex PCR screening method which is used for solving the technical problems in the background technology. The technical scheme of the invention provides a CYP2C19 genotyping multiplex PCR screening method, which comprises the following steps: S1, DNA extraction, namely extracting a target DNA sample from blood by a cleavage method; S2, PCR amplification, namely mixing the DNA sample, the primer, the nuclease, the reaction buffer solution, dNTPs, fluorescent dye and fluorescent marked probe, carrying out PCR amplification, and carrying out electrophoresis detection; s3, placing the PCR reaction solution detected by the S2 into a reaction tube, then placing the reaction tube into a fluorescent quantitative PCR instrument, and collecting fluorescent signals at 75 ℃; S4, judging the result, namely recording the Ct value (marked as CtF) of each detection FAM channel and the Ct value (marked as CtV) of the VIC channel, and calculating the absolute value delta Ct= |CtF-CtV |; If ΔCt <2.5, the locus is judged as heterozygous mutant; If DeltaCt >2.5, it is judged that the locus is judged to be homozygous wild type when CtF < CtV, and homozygous mutant when CtV < CtF; And determining the gene polymorphism condition of the CYP2C19 to be detected through specific values of delta Ct, ctV and CtF, thereby determining whether the reaction of the population carrying the detected gene to the drug is slow metabolism type, fast metabolism type or intermediate metabolism type. In a preferred embodiment, the PCR amplification conditions are 90-95℃pre-denaturation for 15-18min,92-94℃denaturation for 30-35s,62-66℃annealing for 30-35s,70-75℃extension for 1-3min, total of 35-40 cycles, and finally 70-75℃extension for 5-8min, takingThe products were subjected to 3% agarose gel electrophoresis. In a preferred embodiment, the fluorescent dye is selected from one or more of Evagreen, sybrgreen, LC Green Plus. In a preferred embodiment, the fluorescent-labeled probe employs at least one of TAMRA, FAM, HEX, NED, ROX, texas Red, cy5, cy 7. In a preferred embodiment, the sequence of the fluorescently labeled probe matches the wild type sequence of the test gene. In a preferred embodiment, the primers include an upstream primer and a downstream primer. In a preferred embodiment, the fluorescent quantitative PCR instrument is one of LightCycler 480, qiagen Rotor-Gene Q, io-Rad CFX Maestr. The technical scheme of the invention has the beneficial effects that: The detection prevention of the scheme is adopted for detection, the stability of the obtained detection result is high, and the drug substitution condition corresponding to CYP2C19 genotypes in different human bodies can be rapidly and accurately judged so as to facil