Kit and method for detecting gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on Taqman-MGB (Minor Groove Binder) probe

Abstract

The invention belongs to the field of genetic gene detection, and in particular relates to a kit and a method for detecting the gene polymorphism of human MTHFR (methylene tetrahydrofolate reductase) based on a Taqman-MGB (Minor Groove Binder) probe. By adopting the kit and method, two SNP (single nucleotide polymorphism) sites namely C677t and A1298C can be detected at the same time, and the results are easy to judge and read. The MGB probe has a nonluminous 3'-end quenching group and a relatively low background, is more sensitive to detection of a single base mutation, and can judge and read different genotypes only according to a Ct value, and the result can be judged and read more easily compared with that of conventional methods of Taqman probe detection and HRM (high resolution melting) detection. The probe price is relatively low, the detection cost is relatively low, a polymorphism site only needs one-tube qPCR (quantitative polymerase chain reaction) detection, and the operation is simple. Moreover, the method disclosed by the invention does not need subsequent analysis of PCR products, so that while the detection cost is saved, the detection circle is greatly shortened, the detection efficiency is improved and the risk of false positive caused by PCR product pollution is reduced.

Description

technical field [0001] The invention belongs to the field of genetic detection, in particular to a human MTHFR gene polymorphism detection kit and method based on Taqman-MGB probes. Background technique [0002] 5,10-methylenetetrahydrofolate reductase (Mthylenetetrahydrofolate reductase, MTHFR) is a key enzyme in the folate metabolism system, catalyzing the reduction of 5,10-methylenetetrahydrofolate to the methyl donor 5-methyltetrahydrofolate folic acid. On the one hand, 5-methyltetrahydrofolate can be used as an indirect donor of methyl groups to participate in the synthesis of purines and pyrimidines and the methylation of DNA, RNA, and proteins; on the other hand, under the catalysis of methionine synthase, Vitamin B12 is used as a coenzyme to re-methylate homocysteine ​​in the blood to produce methionine, thereby maintaining normal homocysteine ​​levels in the body. There are two common polymorphic sites in the MTHFR gene, C677T (SNP ID: rs1801133) and A1296C (SNP I...

AI Technical Summary

Problems solved by technology

Advantages: accurate results, sequencing method is the gold standard for gene polymorphism site detection; Disadvantages: high cost, cumbersome operation, long test period

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, the operation is cumbersome and the test cycle is long

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: A sample needs to be divided into 2 tubes for PCR testing to detect the genotype of a SNP site, which is troublesome to operate

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

[0011] At present, the Taqman probe method has the following disadvantages: the results of different genotypes are not significantly different, and three genotype controls must be set up for each qPCR test. In addition, the fluorescence quenching of the TaqMan probe is not complete, and the background is high

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