Primers, probes, kit and method for testing human VKORC1 and CYP2C9 gene polymorphisms

Abstract

The invention belongs to the technical field of genetic engineering, and discloses a primer and probe combination for testing human VKORC1 and CYP2C9 gene polymorphisms, a kit containing the primer and probe combination and a fluorescent PCR (polymerase chain reaction) method for testing human VKORC1 and CYP2C9 gene polymorphisms by adopting the primer and probe combination. The invention, which is based on the TaqMan fluorescent PCR technique, is simple, efficient and highly sensitive. In addition, by reasonably matching primers with probes, the interaction between the primers, the interaction between the primers and the probes and the interaction between the probes are effectively prevented, thus reducing testing errors. When used for testing human VKORC1 and CYP2C9 gene polymorphisms, the primers, the probes and the kit which are provided by the invention have the advantages of high sensitivity, high specificity, easiness, quickness and safety in operation, simple and visual result judgment and the like, and can directly use a blood sample or a dried blood spot sample as a template.

Description

technical field [0001] The invention relates to the technical field of genetic engineering, in particular to primers, probes, kits and methods for detecting human VKORC1 and CYP2C9 gene polymorphisms. Background technique [0002] Warfarin is a commonly used anticoagulant drug in clinical practice, and it is the first-line drug for diseases such as deep vein thrombosis, atrial fibrillation, heart valve replacement and pulmonary embolism. However, the therapeutic window of warfarin is very narrow: if the dose is small, it cannot prevent the formation of thrombus; At present, more than 30 genes related to warfarin pharmacokinetics and pharmacodynamics are known, among which vitamin K epoxide reductase complex subunit 1 (VKORC1) and cytochrome P450 2C9 (CytochromeP4502C9 , CYP2C9) gene polymorphism is the main factor influencing the individual dose difference of warfarin. [0003] There are wild-type CYP2C9*1 and mutant CYP2C9*2~CYP2C9*13 in the CYP2C9 gene, among which the m...

AI Technical Summary

Problems solved by technology

Although the sequencing method is the "gold standard" for gene polymorphism detection, it requires a long detection time, complicated operation, and low sensitivity; the PCR-gene chip method has high operational requirements, and hybridization and washing must be protected from light operation, and the chip is expensive; the PCR-melting curve method is non-specific and cannot precisely locate the position of the gene change, and the difference in Tm caused by a single base difference is not very obvious, which usually results in inconspicuous peaks on the melting curve Therefore, there are certain difficulties in the clinical promotion of these three methods

The traditional fluorescent quantitative PCR method is widely used clinically. This method has certain requirements on the number of samples and the distribution of polymorphisms. When the sample size is small, it cannot accurately detect the gene polymorphisms of the samples. It is very useful for detecting human VKORC1 and CYP2C9 gene polymorphisms. However, it is still unable to meet the requirements of high efficiency, precision, and simplicity, and at the same time, it is necessary to perform cumbersome and complicated operation procedures such as genomic DNA extraction on the sample, so there is an urgent need for a fast, effective, accurate detection method that does not require DNA extraction

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