Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene

A gene polymorphism and kit technology, applied in the field of molecular biology, can solve the problems of high cost, time-consuming and laborious, and unsuitable detection, and achieve the effects of simple operation, reduced bleeding risk, and clear genotyping

Pending Publication Date: 2021-02-02
上海利康精准医疗技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0006] At present, the commonly used mutation detection method is direct DNA sequencing method. PCR products can be directly analyzed by DNA sequence, which can clarify the mutation site, but it has the disadvantages of time-consuming, laborious, expensive, and not suitable for detection of a large number of samples.

Method used

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  • Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene
  • Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene
  • Probe, primer and kit for detecting polymorphism of CYP2C9 gene and VKORC1 gene

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0043] Embodiment 1: probe and primer design

[0044] The present invention designs probe and primer sequences for CYP2C9*2C>T, CYP2C9*3A>C, VKORC1-1639G>A SNP sites. The specific principle is to use the conformational change of the fluorescent probe and the target sequence after hybridization to release the fluorescent dye, and judge the genotyping results according to the peak diagrams and Tm values ​​at different temperatures after hybridization. In the absence of target DNA, the fluorophore and quencher can be stably combined together, and no fluorescent signal can be detected; when there is target DNA, the structure of the fluorescently labeled probe is destroyed, and the fluorophore and quencher The extinguishing groups are separated from each other, and the fluorescent signal can be detected.

[0045] Design the probe and primers so that the probe is in a stem-loop state in the absence of the template at the annealing temperature. Take CYP2C9*2 as an example figure 1...

Embodiment 2

[0062] Embodiment 2: Detect different genotype standard products

[0063] 1. Use plasmids CYP2C9*2, CYP2C9*3 and VKORC1 to construct and prepare wild-type standard plasmids and mutant standard plasmids containing the target gene rs1799853, rs1057910, and rs9923231 sites (the source of the plasmid, and the synthesis of the plasmid containing the target gene by Synthesized by Sangon Bioengineering (Shanghai) Co., Ltd. The accuracy of the sequence was determined by Sanger sequencing. The genotype of the wild-type standard plasmid rs1799853 is CC, the genotype of rs1057910 is AA; the genotype of rs9923231 is GG; the mutant standard plasmid rs1799853 The genotype was TT, the genotype of rs1057910 was CC, and the genotype of rs9923231 was AA. The standard plasmid DNA concentration was standardized to 10ng / ul.

[0064] 2. Using the probes and primers in Example 1.

[0065] 3. PCR reaction system:

[0066] 1) Add 7.5ul of PCR Mix, 0.5uM of 3 sets of forward primer solutions and 0.5u...

Embodiment 3

[0069] Example 3: Double-blind experimental investigation using warfarin individualized drug-related genes (CYP2C9*2, CYP2C9*3 and VKORC1) detection kits

[0070] 1. The genomic DNA of oral epithelial cells of 50 cases of healthy volunteers in Shanghai was extracted by silica gel adsorption method, and the concentration and purity of DNA were detected by electrophoresis gel imaging, and the DNA concentration of the samples to be tested was standardized to 10ng / ul.

[0071] 2. The detection method is as follows: Add 7.5ul of PCR Mix, 0.5uM of each forward primer solution, 0.5uM of each reverse primer solution, and 0.1uM of each probe to each PCR reaction well, and carry out a weak positive control (rs1057910 gene rs9923231 genotype is AC and rs9923231 genotype is AG), negative control (rs1057910 genotype is AA, rs1799853 genotype is CC and rs9923231 genotype is GG) and samples to be tested, add 2ul of DNA to each reaction well, and use sterilized weight Make up 15ul with distil...

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Abstract

The invention discloses a probe, primer and kit for detecting polymorphism of a CYP2C9 gene and a VKORC1 gene. The sequence of the probe is shown as SEQ ID NO.: 1, SEQ ID NO.: 4 or SEQ ID NO.: 7, or the two ends of the sequences of SEQ ID NO.: 1, SEQ ID NO.: 4 and SEQ ID NO.: 7 are modified by groups. The probe, primer and kit for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene have the advantages of high sensitivity, good specificity, rapidness, high-throughput detection and the like when being used for detecting the polymorphism of the CYP2C9 gene and the VKORC1 gene.

Description

technical field [0001] The invention relates to the field of molecular biology, in particular to probes, primers and kits for detecting CYP2C9 gene and VKORC1 gene polymorphisms. Background technique [0002] Warfarin is currently the earliest, largest and most widely used oral anticoagulant in clinical practice, and has been used in anticoagulant therapy for many diseases, such as primary and secondary prevention of venous thromboembolic disease (VTE), atrial Prevention of tremor thromboembolism, valvular disease, artificial valve replacement, and intracardiac thrombosis, etc., but the clinical efficacy and adverse reactions vary greatly from individual to individual, and the dose is difficult to control, especially in the initial stage of warfarin anticoagulant therapy, which can easily lead to Serious bleeding complications. It is estimated that bleeding side effects occur in 15.2% of people every year, of which fatal major bleeding accounts for 3.5%, and the difference ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12Q1/6883C12N15/11
CPCC12Q1/6883C12Q2600/106C12Q2600/156
Inventor 毛丹丹张小燕张奕傅咏南
Owner 上海利康精准医疗技术有限公司
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