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Fluorescent quantitative PCR detection method based on CYP2C19 gene

A CYP2C19 and fluorescence quantitative technology, applied in the biological field, can solve the problems of cumbersome operation and difficult to meet the needs of clinical routine detection, and achieve the effect of convenient operation, prevention of adverse reactions, and simple operation

Inactive Publication Date: 2019-02-01
济南星齐医学检验有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Clinical detection methods for CYP2C19 gene polymorphisms include: restriction fragment length polymorphism analysis (PCR-RFLP), allele-specific PCR, DNA sequencing and gene chip technology, etc., but these methods are cumbersome to operate and difficult to meet the routine clinical detection need

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0028] The technical scheme that the present invention adopts to solve its technical problem is: a kind of fluorescent quantitative PCR detection method based on CYP2C19 gene, comprises the following steps:

[0029] (1) Weigh 10 ng of CYP2C19 genomic DNA, add 2 μl of 10×Taq DNA polymerase (without Mg2+), shake the tube body vigorously for 10 seconds, add 0.4 μl of DNA polymerase, incubate at 15°C for 2 minutes, then add 0.2 μl of uracil N-glycosylase, shake and mix for 15 minutes at 0°C;

[0030] Measure 0.1 μl of deoxyribonucleoside triphosphate and 0.2 μl of Mg2+ aqueous solution, mix well, add to the above mixed liquid, vibrate the tube body manually for 10 seconds, and place the mixed liquid in a 60°C metal bath for 3 minutes;

[0031] Measure 2 μl of glycerol, 0.05 μl of downstream primer, 0.05 μl of downstream primer, 0.1 μl of 681G probe, 0.1 μl of 681A probe, add to the above mixed liquid, shake the solution for 10 seconds, and place it in a centrifuge at 6000 rpm for ...

Embodiment 2

[0050] The technical scheme that the present invention adopts to solve its technical problem is: a kind of fluorescent quantitative PCR detection method based on CYP2C19 gene, comprises the following steps:

[0051] (1) Weigh 15 ng of CYP2C19 genomic DNA, add 3 μl of 10×Taq DNA polymerase (without Mg2+), shake the tube body vigorously for 10 seconds, add 0.6 μl of DNA polymerase, incubate at 15°C for 3 minutes, then add 0.3 μl of uracil N-glycosylase, shake and mix for 30 minutes at 0°C;

[0052] Measure 0.15 μl of deoxyribonucleoside triphosphate and 0.3 μl of Mg2+ aqueous solution, mix well, add to the above mixed liquid, vibrate the tube body manually for 15 seconds, and place the mixed liquid in a 70°C metal bath for 3 minutes;

[0053]Measure 3 μl of glycerol, 0.1 μl of downstream primer, 0.1 μl of downstream primer, 0.2 μl of 681G probe, 0.2 μl of 681A probe, add to the above mixed liquid, shake the solution for 15 seconds, and place it in a centrifuge Briefly centrifug...

Embodiment 3

[0072] The technical scheme that the present invention adopts to solve its technical problem is: a kind of fluorescent quantitative PCR detection method based on CYP2C19 gene, comprises the following steps:

[0073] (1) Weigh 20 ng of CYP2C19 genomic DNA, add 4 μl of 10×Taq DNA polymerase (without Mg2+), shake the tube body vigorously for 15 seconds, add 0.8 μl of DNA polymerase, incubate at 30°C for 3 minutes, then add 0.4 μl of uracil N-glycosylase, shake and mix for 30 minutes at 4°C;

[0074] Measure 0.2 μl of deoxyribonucleoside triphosphate and 0.4 μl of Mg2+ aqueous solution, mix well, add to the above mixed liquid, vibrate the tube body manually for 15 seconds, and place the mixed liquid in a 70°C metal bath for 5 minutes;

[0075] Measure 4 μl of glycerol, 0.1 μl of downstream primer, 0.1 μl of downstream primer, 0.2 μl of 681G probe, 0.2 μl of 681A probe, add to the above mixed liquid, shake the solution for 15 seconds, and place it in a centrifuge Briefly centrifug...

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Abstract

The invention relates to a fluorescent quantitative PCR detection method based on CYP2C19 gene, and the method comprises the following steps: weighing 10 ng to 20 ng of CYP2C19 genomic DNA, adding 2 mu l to 4 mu l of 10*Taq DNA polymerase, adding 0.4 mu l to 0.8 mu l of DNA polymerase, then adding uracil N-glycosylase, deoxyribonucleoside triphosphate and a Mg<2+> aqueous solution, measuring 2mu l-4mu l of glycerol, 0.05mu l-0.1mu l of a downstream primer, 0.05mu l-0. 1 mu l of a downstream primer, 0.1 mu l to 0.2 mu l of a 681G probe, and 0.1 mu l to 0.2 mu l of a 681A probe to add to the mixed liquid, and adding a proper amount of double-distilled water. Result determination is as follows: according to specific values of DELTA Ct, CtV and CtF, whether the reaction of people carrying a to-be-detected gene to a medicine is a slow metabolic type, a fast metabolism type or an intermediate metabolism type is determined. The method has the beneficial effects that one PCR reaction does notneed to be set for each genotype, and the operation is simple and convenient; and the method is suitable for two-channel fluorescent quantitative PCR (polymerase chain reaction) instruments, and has the advantages of being specific, sensitive, rapid, convenient to operate and the like.

Description

technical field [0001] The invention relates to the field of biological technology, in particular to a CYP2C19 gene-based fluorescent quantitative PCR detection method. Background technique [0002] Cytochrome oxidase CYP2C19 is involved in the metabolism of drugs such as warfarin, clopidogrel, diazepam, propranolol, and omeprazole, and its genetic polymorphism is an important factor that leads to individual differences in drug efficacy. Clinical detection methods for CYP2C19 gene polymorphisms include: restriction fragment length polymorphism analysis (PCR-RFLP), allele-specific PCR, DNA sequencing and gene chip technology, etc., but these methods are cumbersome to operate and difficult to meet the routine clinical detection need. Fluorescent quantitative PCR detection has the characteristics of high sensitivity, high specificity, simple operation, simple result judgment, etc., which is convenient for clinical routine detection application. Therefore, a simple and accurat...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/686
CPCC12Q1/686C12Q1/6883C12Q2600/106C12Q2563/107C12Q2545/114
Inventor 程世亮
Owner 济南星齐医学检验有限公司
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