Kit and method for detecting ALDH2 polymorphism based on FRET probe melting curve

Abstract

The present invention discloses a kit and a method for detecting ALDH2 polymorphism based on a FRET probe melting curve. The kit comprises a forward primer and a reverse primer for specifically amplifying the gene ALDH2 (rs671) locus, and a pair of fluorescently labeled probes for specifically distinguishing the gene ALDH2 (rs671) polymorphism, wherein the specific sequence of the forward primer is F: 5'-AGCAGACCCTAAATCCCTGG-3, the specific sequence of the reverse primer is R: 5'-CACCAGCAGACCCTCAAG-3', the specific sequence of the sensor probe is 5'-TGCAGGCATACACTGAAGTGAAAACTGT-FAM-3', and the specific sequence of the anchor probe is 5'-ROX-AGTGTGGGACCTGCTGGGGGCT-P-3'. According to the present invention, the advantages of good specificity, high sensitivity, low price, simple operation and avoidance of pollution caused by tune opening operation are provided, and the result can be intuitively and clearly interpreted through the TM value read by the instrument.

Description

technical field [0001] The invention belongs to the field of in vitro nucleic acid detection, and in particular relates to a kit and a method for detecting ALDH2 gene polymorphism based on a FRET probe melting curve method. Background technique [0002] Human acetaldehyde dehydrogenase 2 (ALDH2) is one of the most important enzymes in the pathway of ethanol metabolism, and its coding gene ALDH2 has a high degree of polymorphism. In Asian yellow populations, ALDH2 is the most frequent and most important mutant. Recent studies have shown that ALDH2 can inhibit its carrier's dependence on alcohol to a certain extent, and has a profound relationship with alcoholism, alcoholic liver disease, upper gastrointestinal cancer and gastric cancer caused by alcoholism. [0003] The ALDH2 gene mutation also reduces the activity of acetaldehyde dehydrogenase by more than 10 times, which hinders the metabolic process of alcohol from ethanol → acetaldehyde → acetic acid → water and carbon d...

AI Technical Summary

Problems solved by technology

[0006] Gene polymorphism detection methods have become increasingly mature. At present, there are several widely used technical platforms: the first is the traditional RFLP (restriction fragment length polymorphism) method, which uses specific restriction enzymes to identify specific sequences. Gene polymorphisms can be analyzed by agarose gel electrophoresis results. This method is low in cost and the experiment can be completed with ordinary instruments, but the operation is cumbersome, and the operation of opening the cover is likely to cause aerosol pollution, and incomplete enzyme digestion is often Misjudged as heterozygous, poor specificity

The second method is gene sequencing as the "gold standard". The sequencing results are intuitive and have high specificity. However, because sequencing instruments are expensive, they cannot be widely used.

Based on the Taqman probe amplification curve method, the problem of uncapped contamination can be effectively avoided. At the same time, detection instruments have been popularized in many universities, hospitals and other scientific research and medical institutions. However, the specificity is not ideal, and non-specific signals are easily generated, and the optimization is difficult. High, the test result is greatly affected by the quality of the sample

The use of MGB-modified probes can solve the problem of specificity, but the synthesis cost is high and the synthesis route is limited

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