Primer pair and kit for detecting hepatitis B canceration susceptibility gene polymorphism

Abstract

The invention relates to a primer pair and a reagent kit for detecting hepatitis B canceration susceptibility gene polymorphism, and belongs to the technical field of in vitro nucleic acid detection.The primer pair comprises a forward amplification primer, a reverse amplification primer and a sequencing primer, wherein the 5'-end of the reverse amplification primer is labeled with biotin. The kitcomprises a PCR (Polymerase Chain Reaction) solution containing a forward amplification primer, reverse amplification primer, a sequencing primer, uracil DNA (Deoxyribonucleic Acid) glycosylase, Taqpolymerase and a positive control product. The kit provided by the invention has the advantages of accurate detection result, high specificity, short detection period, and simple operation, and the clinical inspection requirements can be effectively met; in addition, the kit also has the advantages of real-time monitoring of reaction progress, and short reaction time, the PCR product can be used for sequencing on a pyrosequencing instrument after being simply treated, the sensitivity is higher than that of a gold standard method, i.e., a capillary electrophoresis sequencing method, and the kitis more applicable to mutation analysis.

Description

technical field [0001] The invention relates to the technical field of in vitro nucleic acid detection, in particular to a pair of primers and a kit for detecting polymorphisms of susceptibility genes for hepatitis B canceration. Background technique [0002] Liver cancer is the third most lethal malignant tumor in the world, and it is also a common malignant disease in China. About 700,000 people die of liver cancer every year worldwide. Hepatocellular Carcinoma (HCC) is the most common type of liver cancer. According to a survey by the International Agency for Research on Cancer in 2000, 80% of HCC patients are related to heterosexual hepatitis virus and hepatitis C virus. Medical history survey shows that more than 80% of Chinese patients with liver cancer have a history of hepatitis B. According to the data released by the Ministry of Health, China currently has a total of 93 million hepatitis B patients, accounting for more than one-third of the total number of hepati...

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Problems solved by technology

[0013] At present, among the existing publicly reported literatures, only the Chinese patents CN201610773305.5 and CN201610773331.8 applied by our research team in 2016 were used for HLA-DQ and STAT4 genotyping in hospitals, respectively. "standard" (PCR-direct sequencing method) has been innovated and improved; however, the technical solutions involved in the two patents CN201610773305.5 and CN201610773331.8, after the two products were put into the clinical market, especially in large-scale applications in hospitals During the process, it was found that the specificity of the designed primers and kits was not particularly high, and the accuracy of the test results would occasionally fluctuate. It is speculated that there may be adaptability problems between the design of the primers and the environment of the actual molecular diagnostic laboratory in the hospital. In addition, When the two kits are used alone, they often cannot meet the clinical needs of two genotypes of hepatitis B cancer susceptibility, and the two kits need to be used in combination, and there are many operational problems and system cross problems in clinical practice; therefore, the two easy There is still a lot of room for improvement in the combination of the combination of the sensitivity gene kit and the redesign and optimization of primers and the stability of the corresponding PCR system; Fully re-optimized the design of the primers of the two R&D projects, combined the two susceptibility gene detection kits into one, optimized the PCR reaction system of the new kit, and significantly improved the product performance of the new kit

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