Pyrosequencing Diagnosis To Hepatitis B Gene Mutation Caused By Drug Resistance

Now, the main methods of detection of hepatitis B virus resistance mutations based on PCR-based direct sequencing, line probe analysis, restriction fragment length polymorphism analysis and gene chips technology. The methods are based on PCR-based operation, and only YMDD resistant mutants of real time quantitative PCR for qualitative detection technology is used in clinical testing. there is no more comprehensive and satisfactory against a variety of qualitative of resistance gene mutation and quantitative detection of mutation strain method.Pyrosequencing technology has high accuracy, flexibility, repeatability, simple operation and other advantages.It has been widely used in many research fields, especially in the SNP analysis. With the development of pyrosequencing, establish the pyrosequencing detection method of Lamivudine and Adefovir dipivoxil treatment of hepatitis B virus resistance mutations, and to provide the basis for clinical diagnosis of hepatitis B drug resistance and treatment. For gene mutation of DNA polymerase of hepatitis B virus, wild-type and mutant plasmid were constructed as a standard. The gene universal PCR primers and the point mutation pyrosequencing primer were designed by bioinformatics tools, and pyrosequencing method was established for mutation detection. The serum samples of patients were detected, who received lamivudine, adefovir dipivoxil treatment for chronic hepatitis.Four common drug resistance mutations of hepatitis B virus in standard strains and mutant clone were constructed, and the pyrosequencing method of lamivudine, adefovir dipivoxil resistance mutations was established. 68 serum samples of patients with resistant or suspected drug resistance were detected and sequencing. Lamivudine resistance mutations were detected in 32 cases, adefovir resistance mutations in 5 cases, including 20 cases of pyrosequencing of mixed mutations, and Sanger sequencing showed 6 cases.Analysis of clinical data , the rate of lamivudine resistance mutations in treatment group is consistent with the findings of Lok AS, The data showed resistance to lamivudine in the treatment group has a higher incidence of drug resistance rate of one-year 25%, 46% of the biennium. rtM204V / I (ie YMDD) the most common drug resistance, rtL180M mutation rate is low, and rtL180M mutations were associated with rtM204V mutations. Detected in patients treated with adefovir dipivoxil is in a low incidence of drug resistance, rtN236T not detected.Pyrosequencing Quantitative detection of lamivudine, adefovir dipivoxil resistance mutation method was successfully established, and the hepatitis B virus drug resistance mutations in the standard plasmid, dynamically monitor of clinical hepatitis B virus variant strains were constructed, which laid the foundation for guiding rational use of drugs.