HBV耐拉米夫定多聚酶基因变异检测方法的建立  

Establishment of a method for the detection of lamivudine-resistant mutants in the sequence of HBV polymerase gene

作　　者：张伟三[1] 丁静娟[2] 

机构地区：[1]贵阳医学院 [2]贵阳医学院附院感染科,贵州贵阳550004

出　　处：《医学文选》2002年第3期270-273,共4页Anthology of Medicine

摘　　要：目的　建立一种简便、准确、实用的乙型肝炎病毒 ( HBV)耐拉米夫定多聚酶基因变异检测方法。方法　根据已公布的 HBV DNA序列 ,在 HBV多聚酶基因区设计并合成了两对寡核苷酸引物 ,其中一只为错配引物 ;应用巢式错配聚合酶链反应特异性扩增含有 HBV多聚酶基因 YMDD基序的片段 ,扩增产物用限制性内切酶 ( Nde )酶切 ,琼脂糖凝胶电泳 ,观察酶切后的目的片段限制性片段长度多态性 ( RFLP)图谱。部分标本进行 DNA测序。结果　 ( 1 )所建立的巢式错配 PCR- RFLP方法操作简便 ,快速 ,从 DNA提取到酶切后电泳分析仅需要 1 1小时 ;灵敏度高 ,可以检测到 1 0 3 copies/ml的 HBV DNA;特异性强 ,结果准确 ,经 DNA测序证实。 ( 2 )应用该方法对 2 0例长期服用拉米夫定的慢性乙型肝炎患者的系列血清进行了检测 ,发现 YMDD变异者 ( 4 5 % ) 9例 ,YMDD野毒株者 ( 5 5 % ) 1 1例 ,表明该方法可有效地鉴别 HBV YMDD野毒株与变异株。结论　本实验所建立的巢式错配 PCR- RFLP方法简单、敏感、特异 ,适合于一般实验室应用及大规模的人群调查。Objective To establish a simple,accurate and practical method for the detection of lamivudine resistant mutants in the sequence of HBV polymerase gene Methods Two pair of primers were synthesized to amplify approximately 119bp of the polymerase gene of hepatitis B virus(HBV) spanning YMDD motif,where mutations associated with resistance to lamivudine invariably occur.One was misparing primer.Then HBV polymerase gene YMDD motif was amplified by using nested mismatched polymerase chain reaction(PCR).The PCR products were digested with Nde I and subjected to electrophoresis on agarose gels.The patterns of restriction fragment length polymorphism(RFLP) of HBV polymerase gene were distinguished Results 1 It looks that the method was extremely rapid,sensitive,specific and accurate.2 Using this new method,we detected 20 chronic hepatitis B patients following long time of lamivudine therapy,55%(11/20) patients had not find mutations,and 45%(9/20) patients developed mutations in the YMDD motif of HBV polymerase gene.Which could result in amino acid changes.It shows that HBV polymerase gene mutations could be confirmed.The result was confirmed by DNA sequencing Conclusions The nested mismatched PCR RFLF method is considered a rapid and simple,specific and sensitive technique for the detection of lamvudine resistance associated mutations in HBV.This method is suitable for routine laboratories and large scale population studies

关 键 词：聚合酶链反应 限制性片段长度多态性分析 拉米夫定 乙型肝炎病毒 基因变异 耐药 

分 类 号：R346[医药卫生—基础医学]