【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′TTTAAA3′),而变异株(rt236T)无此限制性酶切位点。选取4份应用ADV治疗1年以上出现HBVDNA反跳的临床耐药慢性乙型肝炎患者血清,经PCR扩增、DraⅠ酶切、3%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1例进行HBVRT区基因序列分析。结果自4份血清标本中检测到1例rtN236T变异。所建立的ntPCR-RFLP方法灵敏度高,可以检测到103copies/L的HBVDNA;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rtN236T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。更多还原

【Abstract】 Objective To establish a simple, accurate and practical method for detection of adefovir dipivoxil resistance-associated mutation in hepatitis B virus: rtN236T mutation.Methods Two pair of primers were designed to amplify the region of HBV reverse transcriptase codon 236(rt236) in order to introduce an DraI restriction site upon PCR product of wild-type (wt). The creation of an DraI site occurs only if the template HBV has the wt DNA sequence and is absent if the template HBV is mutated. After amplification, the PCR products were digested with DraI and subjected to electrophoresis. The patterns of restriction fragment length polymorphism (RFLP)of HBV adefovir dipivoxil resistance-associated mutation were distinguished.Results The method was extremely rapid, sensitive, specific and accurate. Of 4 patients with HBV DNA breakthrough during adefovir dipivoxil therapy, 1 rtN236T mutation was detected by this method. The result were confirmed by DNA sequencing.Conclusion The ntPCR-RFLP assay is a rapid, simple, specific and sensitive method for detection of adefovir dipivoxil resistance-associated mutation in hepatitis B virus.更多还原