一种新的HBV DNA rtA181T耐药突变检测方法  

A new method to detect drug-resistant mutation of HBV DNA rtA181T

作　　者：陈洪涛[1] 何桂蓉[2] 吴诗品[1] 

机构地区：[1]暨南大学医学院附属第二临床医学院感染内科,广东深圳518020 [2]暨南大学医学院附属第二临床医学院检验科,广东深圳518020

出　　处：《南京医科大学学报（自然科学版）》2016年第7期880-885,共6页Journal of Nanjing Medical University(Natural Sciences)

基　　金：广东省药学会肝炎用药研究基金(2012G24)

摘　　要：目的 :通过巢式PCR及特异引物,建立一种新的乙肝病毒(HBV)DNA rtA181T耐药突变的PCR直接扩增检测方法。方法:巢式PCR方法第1轮扩增HBV DNA逆转录酶rt活性域片段,第2轮PCR上游引物3′末端碱基设计为与HBV DNA rtA181T突变碱基相同的碱基,扩增出的目的基因片段,即为突变片段。利用该方法 ,本文检测了43例HBV DNA rtA181T变异标本,分析该方法检测的灵敏性;并比较分析低拷贝HBV DNA水平与高拷贝HBV DNA水平下,该方法与PCR-Sanger测序法检测HBV DNA rtA181T突变的一致性。结果:产物经测序证实,突变检测引物巢式PCR法能扩增出HBV DNA rtA181T耐药突变。在HBV DNA水平为24 U/μL、rtA181T突变株含量低至10%时,该方法仍能较好检测出rtA181T变异片段。对43例不同拷贝水平的HBV DNA rtA181T耐药变异临床标本检测显示,该方法检测灵敏性达100%,常规PCR-Sanger测序灵敏性为74%,差异有统计学意义(P<0.05)。结论 :基于巢式PCR及突变引物为基础的HBV DNA rtA181T耐药突变PCR直接检测法能较好地检测HBV DNA rtA181T耐药变异发生;对HBV DNA低拷贝水平下的rtA181T耐药变异检测,该方法灵敏性优于Sanger测序法,且有较好的特异性。这对早期发现HBV DNA耐药突变、及时更改抗HBV治疗策略具有重要临床指导意义。Objective：To construct a new direct amplification PCR method to detect HBV rtA181T drug resistant mutation by nested PCR and specific primers. Methods： Nested PCR was performed to amplify the HBV polymerase rt domain fragment in the first round, and the base in the 3＇ end of the sense primer was designed same as the mutation one of HBV DNA rtAlglT in the second round. Thus, the amplified fragment was the HBV DNA rtA181T mutation fragment. Using this method, we examined 43 specimens of various HBV DNA loading with HBV DNA rtA181T mutation and analyzed the sensitivity of this method; and compared it with the PCR-Sanger sequencing method in consistency under the level of low or high copy DNA HBV. Results： Confirmed lay PCR products sequencing, this nested PCR based specific primers had successfully detected the HBV DNA rtA181T drug resistant mutation. This method had confirmed rtA181T drug resistant mutation even when the virus load was 24 U/μL and the mutation strain was less than 10% in the whole quasi-species pool. By detection of the 43 specimens, we found that the sensitivity of this method was 100%, significantly superior to Sauger sequencing, which was 74% （P 〈 0.05）. Conclusion：Based on nested PCR and specific primers, our new method can successfully detect HBV DNA rtA181T mutation. Especially in low virus load circumstance, this new method is superior to Sanger sequencing in sensitivity, and also shows good specificity, thus is beneficial to early detection of HBV DNA drug resistant mutation and important for adjustment of the anti-HBV therapy in time.

关 键 词：乙型肝炎病毒 耐药性 突变 基因测定 

分 类 号：R512.6[医药卫生—内科学]