应用ntPCR-RFLP技术检测乙型肝炎病毒阿德福韦耐药变异  

Application of nest PCR-RFLP in the detection of adefovir dipivoxil resistance-associated mutation in hepatitis B virus

作　　者：闫杰[1] 谢雯[2] 王磊[3] 冯鑫[2] 宋淑静[2] 成军[2] 于岩岩[1] 

机构地区：[1]北京大学第一医院感染科,北京100034 [2]北京地坛医院,北京100011 [3]山东大学医学院传染病教研室,山东济南250012

出　　处：《中国病原生物学杂志》2008年第1期1-4,共4页Journal of Pathogen Biology

基　　金：首都医学发展科研基金资助项目(No.2002-3046)

摘　　要：目的建立一种简便、快速、实用的乙型肝炎病毒(HBV)阿德福韦(ADV)耐药变异-rtN236T变异及rtA181V变异的快速检测方法。方法根据GenBank收录的HBV基因全序设计巢式PCR引物,使野生株rt236N PCR产物中含有DraⅠ酶切位点(5'-TTTAAA-3'),而变异株rt236T无此限制性酶切位点;使野生株rt181A PCR产物中含有BlpⅠ酶切位点(5'-GCTNAGC-3'),而变异株rt181V无此限制性酶切位点。选取4份应用ADV治疗1年以上出现HBV DNA反跳的临床耐药慢性乙型肝炎患者血清,经PCR扩增、限制性内切酶酶切及凝胶电泳,进行限制性片段长度多态性(RFLP)分析。并选择经该方法鉴定的野生株及变异株各1例进行HBV RT区基因序列分析。结果建立的ntPCR-RFLP方法可以检测到102copies/L的HBV DNA;RFLP分析结果与DNA测序结果一致,4份血清标本中检测到1份rtN236T变异,2份rtA181V变异。结论应用ntPCR-RFLP方法检测HBV阿德福韦耐药变异具有灵敏、特异、简便的优点,适用于HBV耐药变异的监测。Objective To establish a simple, accurate and practical method for detection of adefovir dipivoxil resistanceassociated mutations in hepatitis B virus（HBV） ： rtN236T mutation and rtA181V mutation. Methods Primers were designed to amplify part of HBV reverse transcriptase in order to introduce restriction sites upon PCR product of wild-type （wt）. After PCR reaction, the creation of an Dra Ⅰ site occured only if the template HBV has the wt DNA sequence （rt236N） and was absent if the template HBVis mutated（rt236T）. And a BlpⅠ restriction site was created in rt181A （wt）, whereas the Blp Ⅰ site was absent in the variant sequence（rt181V）. The PCR products were digested with Dra Ⅰ or Blp Ⅰ and subjected to electrophoresis. The patterns of restriction fragment length polymorphism （RFLP） of HBV adefovir dipivoxil resistance-associated mutation were distinguished. Results Of 4 patients with HBV DNA breakthrough during adefovir dipivoxil therapy, 1 rtN236T mutation and 2 rtA181V mutation were detected by this method. The results were confirmed by DNA sequencing. Conclusion The mispairing polymerase chain reaction-restriction fragment length polymorphism （ntPCR-RFLP） assay is a rapid, simple, specific and sensitive method for detection of adefovir dipivoxil resistance-associated mutation in hepatitis B virus.

关 键 词：阿德福韦 肝炎病毒 乙型 变异 巢式聚合酶链反应-限制性片段长度多态技术 

分 类 号：R373.21[医药卫生—病原生物学]