Method for quantitative PCR (polymerase chain reaction) detection on CYP2C9 and VKORC1 gene polymorphism

A technology of gene polymorphism and fluorescence quantification, which is applied in biochemical equipment and methods, microbial measurement/testing, DNA/RNA fragments, etc., can solve problems such as cumbersome operation steps, contamination of amplification products, and long detection cycle. Achieve the effects of improving detection efficiency, high accuracy of results, and improving specificity

Pending Publication Date: 2020-02-21
BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Among them, the most commonly used sequencing method can directly detect the position and type of the mutation site, but this ...

Method used

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  • Method for quantitative PCR (polymerase chain reaction) detection on CYP2C9 and VKORC1 gene polymorphism
  • Method for quantitative PCR (polymerase chain reaction) detection on CYP2C9 and VKORC1 gene polymorphism
  • Method for quantitative PCR (polymerase chain reaction) detection on CYP2C9 and VKORC1 gene polymorphism

Examples

Experimental program
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Embodiment 1

[0037] A method for preparing a DNA sample to detect CYP2C9 and VKO ruler C1 gene polymorphism, comprising the steps of: extracting genomic DNA with blood / cell / tissue genomic DNA extraction kit (DP304), and using NP80-touch (Germany IMPLEN ) to determine the concentration and purity of the DNA, and then preserve the genomic DNA.

Embodiment 2

[0039] 1. Primer and Probe Design

[0040] The present invention designs specific primers and specific fluorescent probes respectively according to the base sequences of CYP2C9*2, CYP2C9*3 and VKORC1 gene loci. The specific primers are designed at both ends of the SNP loci by Primer Premier 5.0. Experimental analysis Select the most suitable primer; the specific probe is located in the region between a pair of primers, where the Tm value should be between 65-70°C, usually 5-10°C higher than that of the primers. The 5' end of the probe for detecting the wild type is labeled with a fluorescent reporter group (FAM), the 5' end of the probe for detecting the mutant type is labeled with a fluorescent reporter group (VIC), and the 3' end is labeled with a non-fluorescent quencher group ( NFQ) label, and the MGB modification group is also linked to the probe; in actual operation, the fluorescent reporter group can be replaced according to the actual situation, as long as the fluoresc...

Embodiment 3

[0066] Embodiment 3: the configuration of kit

[0067] According to the above experimental results, this embodiment provides a preferred kit for detecting CYP2C9 and VKORC1 gene polymorphisms by fluorescent quantitative PCR, which includes the following reagents:

[0068] 1. 2×TaqMan PCR Mix: DNA polymerase, buffer, uracil DNA glycosylase, dNTPs (including dUTP);

[0069] 2. Probe Mix: three groups of fluorescent probes, the final concentration is 0.6μM;

[0070] 3. Primer Mix: three sets of primer pairs, F upstream primer (1.0 μM), R downstream primer (1.0 μM);

[0071] 4. Sample DNA extraction reagents;

[0072] 5. Sample DNA collection storage box (such as including oral swabs and swab storage boxes);

[0073] 6. Ultrapure water.

[0074] The reagents are properly placed in the kit, and then put into the instructions for use of the kit (optionally reconfigure PCR test tubes or orifice plates or pipette guns). The instructions for use include the collection steps of the ...

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Abstract

The invention provides a primer probe combination for quantitative PCR (polymerase chain reaction) detection on CYP2C9 and VKORC1 gene polymorphism. The primer probe combination comprises three groupsof primers and double probes shown in sequence tables in the description. The invention further provides a method for quantitative PCR detection on CYP2C9 and VKORC1 gene polymorphism. CYP2C9 and VKORC1 gene polymorphism is detected by using a method of a real-time fluorescence quantitative PCR Taqman-MGB probe, and in addition, a detection method of double probes of a wild type and a mutant typeis adopted, so that not only is specificity improved, but also the cost is lowered; and the Taqman-MGB probe is prior to a common probe in aspects such as experiment result accuracy, repeatability, specificity and sensitivity. Therefore, the method for quantitative PCR detection on CYP2C9 and VKORC1 gene polymorphism, which is established by the invention, has the advantages of being accuracy inresult, high in specific sensitivity, free of toxin or pollution, low in cost, rapid and efficient, and the like.

Description

technical field [0001] The invention relates to the technical field of gene detection, in particular to a combination of primers and probes and a detection method for detecting CYP2C9 and VKORC1 gene polymorphisms by fluorescent quantitative PCR. Background technique [0002] Warfarin is currently a vitamin K antagonist oral anticoagulant widely used clinically. It is used for anticoagulant therapy and prevention. The warfarin preparation currently used clinically is a racemic mixture of S-warfarin and R-warfarin, in which the anticoagulant activity of S-warfarin is 2.7 to 3.8 times that of R-warfarin. It is mainly metabolized by hepatic cytochrome P450 2C9 (CYP2C9). There are single nucleotide polymorphisms (Single nucleotide polymorphisms, SNPs) in the CYP2C9 gene coding region, among which the more important ones are CYP2C9*2 and CYP2C9*3, and the dose of warfarin required for individuals with these two mutations is significantly reduced, There is also an increased ris...

Claims

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Application Information

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IPC IPC(8): C12Q1/6883C12Q1/6858C12N15/11
CPCC12Q1/6883C12Q1/6858C12Q2600/156C12Q2600/106
Inventor 刁久玲翟瑞雪智慧芳倪君君
Owner BEIJING HARMONY HEALTH MEDICAL DIAGNOSTICS CO LTD
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