Quantitative fluorescent PCR inspection for hepatitis-B virus drug-tolerant gene mutation

Abstract

The present invention relates to the real-time quantitative PCR assay for the Lamivudine resistance mutation of hepatitis b virus. The mutations related with Lamivudine resistance locate in the rtL180M and rtM204V / I mutations of DNA polymerase. This method utilizes the character that the primer cannot elongate correctly during the PCR process with mismatches in the 3'-end, designs the mutation site in the 3'-end of the real-time quantitative PCR primer, chooses proper primer concentrations and Touch-down PCR process, and couples the melting point curve of PCR product containing SyBrGreen I. The existence of mutation in the Lamivudine site can be determined with the real-time quantitative PCR signals and the melting point curve. This invention can detect the DNA mutations in the clinical hepatitis b virus specimens rapidly and accurately, and is also suitable for the detection of other gene mutations.

Description

technical field [0001] The invention relates to a fluorescence quantitative touchdown PCR detection method for rapidly detecting the mutation of the hepatitis B virus lamivudine drug-resistant gene, and belongs to the field of biotechnology. Background technique [0002] Hepatitis is a worldwide infectious disease. my country is recognized as a major hepatitis country. The prevalence and incidence of hepatitis are among the highest in the world, especially hepatitis B, which is the most widely distributed nationwide. Hepatitis virus infection is not only closely related to acute and chronic hepatitis, but also closely related to the occurrence of liver cirrhosis and liver cancer. my country is a high-incidence area of ​​HBV infection, about 50%-70% of the population has been infected with hepatitis B, 8%-12% of the population is HBsAg carriers, as many as 100 million people. In recent years, hepatitis B vaccination has played an important role in reducing hepatitis B virus ...

AI Technical Summary

Problems solved by technology

The above-mentioned methods are all based on the operation on the basis of PCR. The gene chip technology is cumbersome to operate and there is inevitably a false positive problem in the hybridization process, and the accuracy is poor; restriction fragment length polymorphism is based on PCR (polymerase chain reaction). Enzyme digestion treatment is performed on the product, which requires that the PCR product should be sufficiently digested for use, and at the same time, the enzyme digestion product must be identified by agarose gel electrophoresis, and the detection sensitivity is greatly reduced

Sequence analysis can accurately identify gene mutations, but there is a bottleneck in the detection of mixed infection strains

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