Method and reagent kit for simultaneously detecting resistance site of three nucleotide analogues of hepatitis B virus

Abstract

The invention provides a method and a reagent kit for simultaneously detecting the resistance sites of the three nucleotide analogues of a hepatitis B virus (HBV). Based on the genotype sequence of the HBV, four nested amplification primers and twenty-seven wild resistance-detection oligonucleotides probes aiming at ten resistance sites are designed in an HBV polymerase area, digoxin-labeled oligonucleotides universal primers are used for conducting nested PRC reaction to amplify a target DNA segment, a target DNA amplification product to be labeled is used for hybridizing with specific oligonucleotide probes on matrix, and the existence of the resistance of the HBV to the three nucleotide analogues is judged through enzymatic color development reaction link-coupled by hybridization conjugates. The method improves the accuracy, the reliability and the sensitivity and can simultaneously detect the ten resistance sites of the three nucleotide analogues, thereby realizing the high-throughput, multi-site, economic and rapid detection and fitting to clinical needs in a better way. The method is of great significance to the realization of early detection and the proper guide of clinical personalized medication.

Description

technical field [0001] The invention relates to a method for detecting drug resistance of hepatitis B virus, in particular to a method for detecting drug resistance of hepatitis B virus to three types of nucleotide analogues by reverse dot hybridization technology, and also relates to a kit for clinical testing. Background technique [0002] Hepatitis B virus infection is widespread worldwide, especially in my country, which accounts for 1 / 3 of the world's infected people. 5%-10% of chronically infected patients can lead to liver cancer (HCC), 30% progress to liver cirrhosis, and 23% of patients with liver cirrhosis will develop liver failure within 5 years. According to statistics, the annual medical and health care expenses for HBV-infected patients in my country are as high as 100 billion RMB. Therefore, HBV infection seriously endangers people's health and causes serious losses to the national economy. [0003] Anti-HBV drug therapy has been recognized internationally ...

AI Technical Summary

Problems solved by technology

Although its detection sensitivity is high, since each detection site needs to establish an independent enzyme cleavage reaction system, the operation is cumbersome, and not every drug resistance site can find the corresponding enzyme cleavage site, which is technically difficult

[0008] (3) Single-strand conformational polymorphism analysis (SSCP): When monitoring the emergence and dynamic changes of drug-resistant strains of viruses, mutations of single bases can be detected, but the amplified PCR products should not be too long, preferably in About 150bp, and if the point mutation occurs at both ends of the amplified fragment, the spatial conformation caused by the point mutation is very small, resulting in almost the same electrophoretic mobility, and false negative results are prone to occur

[0009] (4) Real-time fluorescent PCR: detect HBV mutants with different drug-resistant sites by different melting temperatures, but it is difficult to analyze multiple sites or adjacent sites

[0010] (5) Gene chip technology: high-throughput, multi-site detection of drug-resistant mutations of HBV is possible, but the detection of amplified products requires special instruments, well-trained technicians and multi-step washing and incubation steps, and the process is complicated and time-consuming

These detection methods are fast and accurate, but require expensive instruments, high work intensity, high technical requirements for operators, and are not suitable for mass screening, and are only used for laboratory analysis

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