A kit for rapidly detecting human cyp2c19 gene polymorphism and its application method

Abstract

The invention belongs to the gene detecting technology in clinical detecting technologies of the bio-medical field and particularly relates to a kit for quickly detecting polymorphism of a human CYP2C19 gene and a using method of the kit. The kit comprises three pairs of primers, three probes, six PNA (pentose nucleic acid) sequences, a pair of beta-Actin interior label primers and an interior label probe, and further comprises a PCR (polymerase chain reaction) buffer solution of Mg<2+>, a dNTP mixture, Taq enzyme and ddH2O. The kit can be utilized to carry out qualitative detection on a polymorphic site of the CYP2C19 gene and carry out two-tube PNA-PCR fluorescent amplified reaction according to the SNP site and can carry out result judgment according to a condition whether a two-tube fluorescence curve is started or not without producing personal errors, so that a false positive rate and a false negative rate are low. And moreover, the kit can be used to realize high-flux detection easily and is capable of clinical use.

Description

technical field [0001] The invention belongs to the gene detection technology in the clinical detection technology in the field of biomedicine, and specifically relates to a kit for rapidly detecting human CYP2C19 gene polymorphism and a use method thereof. Background technique [0002] Cytochrome P450 (Cytochrome P450, CYP450) is an isoenzyme encoded by a group of structurally and functionally related superfamily genes (Superfamilygene). For humans, it mainly exists in liver cell microsomes. In the CYP450 superfamily, CYP2 is the largest family, with 15 subfamilies (CYP2A to 2Q), which metabolize about 20% of the drugs currently used clinically, among which CYP2A, CYP2C, CYP2D and CYP2E all have genetic polymorphisms and races difference. CYP2C accounts for about 20% in human liver, and CYP2C8, 2C9 / 10, 2C18 and 2C19 have been cloned. In recent years, the research on the correlation between the genotype of CYP450 and the activity of drug-metabolizing enzymes in different i...

AI Technical Summary

Problems solved by technology

Advantages: low cost, intuitive results; Disadvantages: Some polymorphic sites do not have suitable restriction enzyme sites, and mutations need to be introduced manually. In addition, it is easy to cause product contamination, resulting in false positive results. At the same time, the operation is cumbersome, Long test cycle

Disadvantages: PCR products need to be analyzed later, and the operation is cumbersome; in addition, the results are not easy to interpret, and false positives are prone to occur

Disadvantages: It is difficult to design primers, and when there are a large number of GC or AT sequences at polymorphic sites, it is often helpless, and it is difficult to judge the results

Advantages: One SNP site genotype only needs one tube of PCR to complete the detection; Disadvantages: It is not easy to interpret the difference in Tm value according to the HRM melting curve, and the difference in the peak diagram of the melting curve of different genotypes is not obvious

Disadvantages: Taqman probes need to use MGB-Taqman probes. When the upstream and downstream of the SNP site contain a large number of GC or AT bases, the design and synthesis of MGB-Taqman probes are very difficult and expensive

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