ntPCR-RFLP检测HBV阿德福韦耐药变异-rtA181V变异

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

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- 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(rt181 A)PCR产物中含有Blp Ⅰ酶切位点(5’GCTNAGC3’),而变异株(rt181V)无此限制性酶切位点．选取4份应用ADV治疗1 a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、Blp Ⅰ酶切、30 g／L琼脂糖凝胶电泳,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一例进行 HBV RT区基因序列分析及对照质粒的构建．结果:自4份血清标本中检测到2例rtA181V变异．所建立的ntPCR—RFLP方法灵敏度高,可以检测到103 copies／L的HBV DNA;特异性强,其 RFLP分析结果与DNA测序结果一致．结论:应用ntPCR-RFLP方法检测rtA181V 变异具有灵敏、特异、简便的优点,适用于 ADV耐药变异的临床监测工作．

AIM： To establish a simple, accurate and practical method for the detection of adefovir dipivoxil resistance-associated mutation （rtA181V mutation） in hepatitis B virus. METHODS： Four shares of serum were collected from patients who had been treated with adefovir dipivoxil for more than 1 year, and HBV DNA replication reoccurred. Two pairs of primers were designed to amplify the region of HBV reverse transcriptase codon 181（rt181） in order to introduce a BlpI restriction site upon PCR product of wild type （wt）. After amplification, the PCR products were digested with BlpI and then subjected to electrophoresis. The patterns of rtA181V mutation were detected by restriction fragment length polymorphism （RFLP） analysis. RESULTS： Of the 4 patients, rtA181V mutation was detected in 2 cases by the above method. The established nest PCR-RFLP assay could detect HBV DNA at the level of 103 copies/L. The result of RFLP analysis was in accordance with that of DNA sequencing. CONCLUSION： The ntPCR-RFLP assay is a rapid, simple, specific and sensitive method for the detection of rtA181V mutation in HBV.