CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit

Abstract

The invention discloses a CYP2C19, CYP2C9 and VKORC1 genotyping multiplex amplification system and detection kit. The amplification system comprises two forward primers and a reverse fluorescent primer of six SNPs (single nucleotide polymorphisms) respectively and can simultaneously amplify the six SNPs. The system is characterized by achieving one-tube amplification of three SNPs on the gene CYP2C19, two SNPs on the gene CYP2C9 and one SNP on the gene VKORC1 through multiple PCR (polymerase chain reaction). In particular, the amplification system can achieve direct amplification of blood and blood spot samples and dispenses with the step of extracting DNA (deoxyribonucleic acid). The amplification system can integrate the UDG-dUTP antipollution measure and can effectively prevent product pollution. The detection system is comprehensive in site detection, is simple and convenient to operate, has high specificity, high sensitivity and strong reliability, is low in cost and has the capacity of mass detection.

Description

technical field [0001] The invention relates to a simultaneous detection of CYP2C19, CYP2C9 and VKORC1 gene polymorphisms related to the drug metabolism rate of clopidogrel and warfarin based on multiple fluorescent PCR technology and capillary electrophoresis technology, which is an individualized method for clinical treatment of cardiovascular and other diseases It provides reference in medication and belongs to clinical molecular detection technology in the field of biomedicine. Background technique [0002] Cytochrome P450 superfamily (Cytochrome P450proteins, CYP) is a group of isoenzymes encoded by superfamily genes related to structure and function, which mainly exist in monooxygenases in liver and intestine. Free in the cytoplasm in prokaryotes, it is a soluble protein; in eukaryotes, as a membrane-bound protein, it is mainly distributed on the inner membrane of the endoplasmic reticulum, microsomes and mitochondria, and its main function is in the drug ( Such as cl...

AI Technical Summary

Problems solved by technology

The main disadvantages of this method are: (1) Before the PCR reaction, DNA needs to be extracted from the sample to detect the quality and concentration, the operation is complicated, and there is a risk of confusing the sample

(2) When detecting multiple sites, one sample needs to perform multiple reactions, the detection cost is high, the demand for the amount of template is high, the operation intensity is high, and it is easy to cause operation errors and pollution

Generally speaking, the steps are cumbersome, the test period is long, and the cost is high, which is not conducive to large-scale clinical promotion

2. RFLP analysis method: Based on the change of the restriction endonuclease recognition site caused by gene mutation, such as the loss of the site or the generation of a new site, a specific fragment is amplified by PCR, and then the restriction endonuclease is used for enzymatic analysis. Cutting reaction, electrophoresis to observe the size of the fragments, but it has limitations of enzyme cutting sites, and it is difficult to perform high-throughput parallel analysis

3. Fluorescent quantitative PCR: Real-time fluorescent quantitative PCR has the advantages of simple and fast operation, high experimental sensitivity, quick and quantifiable results, etc. However, this technology has the advantages of easy sample contamination, cross-reaction, and high false positive rate

It can perform high-throughput detection, but this method is expensive, requires a large amount of templates, and the operation and data analysis are complex and time-consuming

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