Multiple PCR screening method for CYP2C19 genotyping

Abstract

The invention discloses a multiple PCR screening method for CYP2C19 genotyping. The multiple PCR screening method comprises the following steps: DNA extraction: extracting a target DNA sample from blood through a lysis method; pCR amplification: mixing the DNA sample, a primer, nuclease, a reaction buffer solution, dNTPs, a fluorescent dye and a fluorescence labeling probe, carrying out PCR amplification, and carrying out electrophoresis detection; s2, putting the PCR reaction solution detected in S2 into a reaction tube, then putting the reaction tube into a fluorescent quantitative PCR instrument, and collecting a fluorescent signal at 75 DEG C; and judging a result. According to the scheme, the detection method is adopted for detection, the obtained detection result is high in stability, and the drug agency conditions corresponding to CYP2C19 genotypes in different human bodies can be rapidly and accurately judged, so that subsequent treatment is facilitated. Through setting of PCR amplification parameters in the scheme, the detection accuracy can be improved, meanwhile, an amplification result test is carried out after amplification, it is guaranteed that all products entering a tester are successfully amplified, and the experiment efficiency and accuracy are improved.

Description

Technical Field

[0001] This invention relates to the field of CYP2C19 genotyping screening technology, and more specifically, to a CYP2C19 genotyping multiplex PCR screening method. Background Technology

[0002] CYP2C19 genotyping refers to determining an individual's type of drug metabolism ability by analyzing specific loci of the CYP2C19 gene. Knowing a patient's CYP2C19 genotype allows for optimized drug dosage adjustments, thereby improving drug efficacy and reducing the risk of adverse reactions. The enzyme encoded by the CYP2C19 gene is involved in the metabolism of many drugs in the liver, including clozapine, clonazepam, chlorpromazine, chlorzoxazone, and tramadol. Based on different CYP2C19 genotypes, individuals can be classified into the following drug metabolism types: Normal metabolizers: These individuals carry two normally functioning CYP2C19 alleles and metabolize drugs at a normal rate.

[0003] Slow metabolizers: These individuals typically have both CYP2C19 alleles that are nonfunctional or absent, resulting in significantly reduced or completely absent CYP2C19 enzyme activity. These individuals metabolize some medications more slowly and may require dose reduction or consideration of other treatment options.

[0004] Rapid metabolizers: These individuals typically carry multiple functional copies of the CYP2C19 allele, leading to increased CYP2C19 enzyme activity. This can result in the rapid metabolism of some drugs, requiring higher doses to achieve effective therapeutic levels.

[0005] Intermediate metabolizers: These individuals carry one normally functioning CYP2C19 allele and one non-functional or reduced-functioning allele. Their drug metabolism rate falls between that of normal metabolizers and slow metabolizers.

[0006] The CYP2C19 genotype can be determined using molecular biology techniques such as PCR and sequencing. This genotypic information is crucial for personalized medicine, helping doctors optimize treatment plans, avoid side effects, and improve treatment outcomes. Therefore, establishing PCR screening methods to detect the CYP2C19 genotype to assess a patient's metabolic status is essential. Summary of the Invention

[0007] The purpose of this invention is to provide a CYP2C19 genotyping multiplex PCR screening method to solve the technical problems existing in the background art.

[0008] The present invention provides a CYP2C19 genotyping multiplex PCR screening method, comprising the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

[0009] In a preferred embodiment, the PCR amplification conditions are as follows: pre-denaturation at 90-95℃ for 15-18 min, denaturation at 92-94℃ for 30-35 s, annealing at 62-66℃ for 30-35 s, extension at 70-75℃ for 1-3 min, for a total of 35-40 cycles, followed by a final extension at 70-75℃ for 5-8 min. The product was detected by 3% agarose gel electrophoresis.

[0010] In a preferred embodiment, the fluorescent dye is selected from one or more of Evagreen, Sybrgreen, LC Green, and LCGreen Plus.

[0011] In a preferred embodiment, the fluorescently labeled probe is at least one of TAMRA, FAM, HEX, NED, ROX, TexasRed, Cy5, and Cy7.

[0012] In a preferred embodiment, the sequence of the fluorescently labeled probe matches the wild-type sequence of the gene to be tested.

[0013] In a preferred embodiment, the primers include an upstream primer and a downstream primer.

[0014] In a preferred embodiment, the real-time PCR instrument is one of LightCycler 480, QiagenRotor-Gene Q, or io-Rad CFX Maestr.

[0015] The beneficial effects of the technical solution of this invention are: The detection method employed in this protocol yields highly stable results, enabling rapid and accurate identification of drug metabolism corresponding to different CYP2C19 genotypes in individuals, thus facilitating subsequent treatment. The PCR amplification parameters in this protocol can be optimized to improve detection accuracy. Furthermore, post-amplification testing ensures that all products entering the instrument are successfully amplified, enhancing both experimental efficiency and accuracy. Detailed Implementation

[0016] The present invention will now be described in further detail. The embodiments of the invention are given for the purpose of illustration and description, and are not intended to be exhaustive or to limit the invention to the forms disclosed. Many modifications and variations will be apparent to those skilled in the art. The embodiments were chosen and described to better illustrate the principles and practical application of the invention, and to enable those skilled in the art to understand the invention and design various embodiments with various modifications suitable for a particular purpose. Example

[0017] The present invention provides a CYP2C19 genotyping multiplex PCR screening method, comprising the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

[0018] PCR amplification conditions were: 90℃ pre-denaturation for 15 min, 92℃ denaturation for 30 s, 62℃ annealing for 30-35 s, 70℃ extension for 1 min, for a total of 35 cycles, with a final extension at 70℃ for 5 min. The product was detected by 3% agarose gel electrophoresis.

[0019] The fluorescent dye used was selected from Evagreen. The fluorescently labeled probe was TAMRA. The sequence of the fluorescently labeled probe matched the wild-type sequence of the gene to be tested. The primers included an upstream primer and a downstream primer. The real-time PCR instrument used was a LightCycler 480. Example

[0020] The present invention provides a CYP2C19 genotyping multiplex PCR screening method, comprising the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

[0021] PCR amplification conditions were: 95℃ pre-denaturation for 18 min, 94℃ denaturation for 35 s, 66℃ annealing for 35 s, 75℃ extension for 3 min, for a total of 40 cycles, with a final extension at 75℃ for 8 min. The product was detected by 3% agarose gel electrophoresis.

[0022] The fluorescent dye used is Sybrgreen. The fluorescently labeled probe uses FAM (Fluorescent Atom). The sequence of the fluorescently labeled probe matches the wild-type sequence of the gene to be tested. The primers include an upstream primer and a downstream primer.

[0023] The real-time PCR instrument used was the Qiagen Rotor-Gene Q. Example

[0024] The present invention provides a CYP2C19 genotyping multiplex PCR screening method, comprising the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

[0025] PCR amplification conditions were: 92℃ pre-denaturation for 16 min, 93℃ denaturation for 32 s, 65℃ annealing for 34 s, 70℃ extension for 2 min, for a total of 38 cycles, with a final extension at 72℃ for 6 min. The product was detected by 3% agarose gel electrophoresis.

[0026] The fluorescent dye is selected from LC Green Plus. The fluorescently labeled probe uses at least one of Cy5. The sequence of the fluorescently labeled probe matches the wild-type sequence of the gene to be tested. The primers include an upstream primer and a downstream primer. The quantitative PCR instrument is an io-Rad CFX Maestr. Example

[0027] The present invention provides a CYP2C19 genotyping multiplex PCR screening method, comprising the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

[0028] PCR amplification conditions were as follows: 90-95℃ pre-denaturation for 15-18 min, 92-94℃ denaturation for 30-35 s, 62-66℃ annealing for 30-35 s, 70-75℃ extension for 1-3 min, for a total of 35-40 cycles, with a final extension at 70-75℃ for 5-8 min. The product was detected by 3% agarose gel electrophoresis.

[0029] The fluorescent dye used was selected from LC Green. The fluorescently labeled probe was Texas Red. The sequence of the fluorescently labeled probe matched the wild-type sequence of the gene to be tested. The primers included an upstream primer and a downstream primer. The real-time PCR instrument used was a LightCycler 480.

[0030] Using the screening methods described in Examples 1-4 above, 10 randomly selected human blood samples were tested. Each sample was tested using the methods described in Examples 1-4 respectively, and the metabolic type was determined. The test results are as follows:

[0031] As shown in Table 1 above, the detection results obtained using this protocol are highly stable and can quickly and accurately determine the drug metabolism corresponding to different CYP2C19 genotypes in individuals, facilitating subsequent treatment. The PCR amplification parameters in this protocol can improve detection accuracy. Furthermore, post-amplification testing ensures that all products entering the instrument are successfully amplified, thus improving experimental efficiency and accuracy.

[0032] Obviously, the described embodiments are only a part of the embodiments of the present invention, and not all of them. All other embodiments obtained by those skilled in the art and related fields based on the embodiments of the present invention without inventive effort should fall within the scope of protection of the present invention. Structures, devices, and operating methods not specifically described and explained in the present invention, unless otherwise specified or limited, shall be implemented according to conventional means in the art.

Claims

1. A CYP2C19 genotyping multiplex PCR screening method, characterized in that, Includes the following steps: S1: DNA extraction: Extracting target DNA samples from blood using a lysis method; S2: PCR amplification: The DNA sample, primers, nuclease, reaction buffer, dNTPs, fluorescent dye and fluorescently labeled probe are mixed, PCR amplification is performed, and electrophoresis detection is performed; S3: Place the PCR reaction solution after S2 detection in a reaction tube, then place it in a real-time PCR instrument and collect the fluorescence signal at 75℃; S4: Result Judgment: Record the Ct value (denoted as CtF) of each detected FAM channel and the Ct value (denoted as CtV) of the VIC channel, and calculate the absolute value of the Ct difference ΔCt=|CtF-CtV|; If ΔCt < 2.5, the site is identified as a heterozygous mutant. If ΔCt > 2.5, there are two cases to determine the type: when CtF < CtV, the locus is determined to be homozygous wild type; when CtV < CtF, the locus is determined to be homozygous mutant type. By using the specific values ​​of ΔCt, CtV, and CtF, the gene polymorphism of the tested CYP2C19 can be determined, thereby determining whether the population carrying the tested gene responds to the drug as a slow metabolizer, a fast metabolizer, or an intermediate metabolizer.

2. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, PCR amplification conditions were as follows: 90-95℃ pre-denaturation for 15-18 min, 92-94℃ denaturation for 30-35 s, 62-66℃ annealing for 30-35 s, 70-75℃ extension for 1-3 min, for a total of 35-40 cycles, with a final extension at 70-75℃ for 5-8 min. The product was detected by 3% agarose gel electrophoresis.

3. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, The fluorescent dye is selected from one or more of Evagreen, Sybrgreen, LC Green, and LC Green Plus.

4. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, The fluorescently labeled probe is at least one of TAMRA, FAM, HEX, NED, ROX, Texas Red, Cy5, and Cy7.

5. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, The sequence of the fluorescently labeled probe matches the wild-type sequence of the gene to be tested.

6. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, The primers include an upstream primer and a downstream primer.

7. The CYP2C19 genotyping multiplex PCR screening method according to claim 1, characterized in that, The real-time PCR instrument is one of the following: LightCycler 480, Qiagen Rotor-Gene Q, or io-Rad CFX Maestr.

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