| 汪钦1 郭晏海1 李勇年2 刘永兰1 包晗1 赵锦荣1 梁平1 雷小英1
张菊1 颜真1.乙肝治疗耐药基因突变的焦磷酸测序技术诊断[J].,2011,11(10):1873-1876 |
| 乙肝治疗耐药基因突变的焦磷酸测序技术诊断 |
| Pyrosequencing Diagnosis to Hepatitis B Gene MutationCaused by Drug Resistance |
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| DOI： |
| 中文关键词: 乙肝 耐药基因 焦磷酸测序 拉米夫定 阿德福韦酯 |
| 英文关键词: Hepatitis B Resistance gene Pyrosequencing Lamivudine Adefovir dipivoxil |
| 基金项目:“十一五”国家“艾滋病和病毒性肝炎等重大传染病防治”科技重大专项基金资助（2009ZX10004-311），陕西省科技
攻关项目（2008K09-09），西安市科技计划项目（SF09027）资助。 |
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| 中文摘要: |
| 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。 |
| 英文摘要: |
| Objective: 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 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. 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. |
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