论文部分内容阅读
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 establish a new direct PCR detection method for rtA181T resistance mutation of hepatitis B virus (HBV) DNA by using nested PCR and specific primers. Methods: In the first round of nested PCR, the HBV DNA reverse transcriptase rt activity domain fragment was amplified. The 3 ’end of the second PCR upstream primer was designed to be the same base as the HBV DNA rtA181T mutation base. The purpose of the gene fragment, is the mutation fragment. Using this method, 43 HBV DNA rtA181T variants were detected and the sensitivity of the method was tested. The HBV DNA level was detected by PCR-Sanger sequencing with low copy HBV DNA level and high copy HBV DNA level. rtA181T Mutation Consistency. Results: The product was confirmed by sequencing, and the mutation detection primer nested PCR method can amplify the HBV DNA rtA181T resistance mutation. The method still better detected the rtA181T variant when HBV DNA level was 24 U / μL and the rtA181T mutant content was as low as 10%. The detection results of 43 clinical samples with rtA181T resistance mutation of HBV DNA at different copy levels showed that the sensitivity of this method was 100%, and the sensitivity of PCR-Sanger sequencing was 74%. The difference was statistically significant (P <0.05). Conclusion: The direct detection of HBV rtA181T mutation based on nested PCR and mutation primers can detect the variation of rtA181T resistance in HBV DNA. The detection of rtA181T resistance mutation at low copy level of HBV DNA The method is more sensitive than the Sanger sequencing method and has good specificity. This early detection of HBV DNA resistance mutations, timely change of anti-HBV treatment strategy has important clinical significance.