乙肝治疗耐药基因突变的焦磷酸测序技术诊断

目的：建立焦磷酸测序技术检测拉米夫定和阿德福韦酯治疗乙肝所致乙肝病毒基因耐药突变的定量检测方法，为临床乙肝耐药诊断和治疗提供依据。方法：针对乙肝病毒DNA聚合酶基因序列上4个常见基因突变位点的6种突变形式，分别克隆构建野生型和突变型质粒作为标准品，应用生物信息学手段设计目标基因通用PCR引物和各突变点的焦磷酸测序引物，建立焦磷酸测序的突变检测方法。对接受拉米夫定、阿德福韦酯治疗的慢性乙型肝炎患者血清标本进行检测。结果：构建了乙肝病毒四种常见耐药性突变的标准株和变异株克隆，建立了分别或同时检测拉米夫定、阿德福韦酯耐药突变的焦磷酸测序方法，对68例临床耐药或疑似耐药的患者血清标本进行检测，双脱氧测序验证，检出拉米夫定耐药突变32例，阿德福韦酯耐药突变5例，其中焦磷酸测序检出20例为混合突变，而双脱氧测序显示为6例。结论：成功建立了焦磷酸测序定量检测拉米夫定、阿德福韦酯耐药基因突变的方法，构建了乙肝病毒耐药基因突变的标准质粒，为临床动态监测乙肝病毒变异病毒株、指导合理用药奠定了基础。

Pyrosequencing Diagnosis to Hepatitis B Gene Mutation Caused by Drug Resistance

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. Methods： For gene mutation ofDNA 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. Results： 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. Conclusion： 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.