乙型肝炎病毒耐拉米夫定多聚酶基因变异检测方法研究  

A study on detection method of lamivudine related mutations in hepatitis B virus polymerase gene

作　　者：丁静娟[1] 张伟三[1] 张莉莎[1] 

机构地区：[1]贵阳医学院附院感染病科,550004

出　　处：《中华实验和临床病毒学杂志》2004年第1期24-27,共4页Chinese Journal of Experimental and Clinical Virology

基　　金：贵州省教委科研基金资助 (F2 0 0 0 4)

摘　　要：目的　建立简便、准确、实用的检测乙型肝炎病毒耐拉米夫定多聚酶 (P)基因变异的方法。方法　根据HBV基因序列 ,设计 5只寡核苷酸引物 ,用巢式聚合酶链反应 (nestedPCR)分别扩增HBVP基因B区和C区片段 ,产物用NdeⅠ或NIaⅢ酶切 ,琼脂糖胶电泳 ,分析酶切产物长度多态性(RFLP) ,建立检测P基因变异的方法。对 30例长期服用拉米夫定的慢性乙型肝炎 (慢乙肝 )患者检测YMDD基序及 5 2 6位点变异 ,16例未用拉米夫定的慢乙肝患者为对照。 4份PCR产物作克隆测序以验证方法的准确、可靠。结果　所建的巢式PCR RFLP方法操作简便、快速 ,从模板提取到酶切后电泳分析仅需 11h ;灵敏度高 ,可检测到 10 3拷贝 ml的HBVDNA ;结果准确 ,4份经酶切分别判断为野毒株或变异株的标本经测序证实。 30例用拉米夫定的慢乙肝患者中 ,发现单纯YMDD变异 8例(2 6 7% ) ,YMDD联合L5 2 6M变异 3例 (10 0 % ) ,16例对照未检出上述位点的变异。结论　本方法简便、准确 ,适合较大样本检测。可用于临床筛检常见拉米夫定耐药性HBVP基因变异。Objective To establish a simple and accurate method for rapid detection of lamivudine related mutations in hepatitis B virus (HBV) polymerase gene. Methods HBV polymerase gene fragments of covering B and C active region were amplified by nested polymerase chain reaction (nPCR) or nested mismatched PCR. The PCR products were digested with Nde Ⅰor NIa Ⅲ and subjected to electrophoresis on agarose gel,respectively. The patterns of restriction fragment length polymorphism (RFLP) were distinguished. Using this method,thirty patients with chronic hepatitis B and treated with lamivudine for at least one year were analysed for the lamivudine related mutations in polymerase gene. Sixteen cases without lamivudine therapy were used as controls. Some of the patients were also analysed by clone sequencing. Results The nested mismatched PCR-RFLP method was simple,accurate and rapid. The whole experiments could be finished in eleven hours. The least titers of HBV DNA which could be detected was 10 3 copies/ml. The wild or mutant strains judged by RFLP were identified by clone sequencing. Mutation in the tyrosine-methionine-aspartic-aspartic acid (YMDD) motif of HBV polymerase gene was found in eight patients and mutations of YMDD motif associated with L526M were found in another three patients. However,there were no such mutations in the control cases. Conclusions The nested PCR-RFLP is considered as a simple and accurate method for rapid detecion of lamivudine related mutations in HBV polymerase gene. It is suitable for larger number of sample detection.

关 键 词：乙型肝炎病毒 拉米夫定 多聚酶 基因变异 检测 耐药性 

分 类 号：R346[医药卫生—基础医学]