【摘要】 目的建立乙型肝炎病毒YMDD突变的下一代测序法,并对此方法进行性能评价。方法采用离心柱法提取血清标本中的DNA,设计引物对乙型肝炎病毒P基因区扩增并靶向捕获,通过下一代测序技术对捕获的DNA进行检测,利用生物信息学分析软件系统对检测结果进行分析,得到基因突变结果,建立下一代测序用于乙型肝炎病毒YMDD突变的检测方法。收集慢性乙型肝炎患者血清样本共229例,采用下一代测序法和Sanger测序法同时检测YMDD基因突变,评价下一代测序法的检测性能。选取5例基因突变阳性的样本(包含不同基因突变型别)、2例基因突变阴性样本,采用下一代测序法对所选样本进行重复检测,评价该方法的重复性。结果建立了乙型肝炎病毒YMDD突变的下一代测序检测方法。对229份临床样本的检测中,2种测序方法均检测到74例(74/229,32.31%)YMDD突变,其中下一代测序法检测到44例(44/229,19.21%)rtM204V突变,25例(25/229,10.92%)rtM204I突变,5例(5/229,2.18%)rtM204V/I混合突变。Sanger测序法检测到47例(47/229,20.52%)rtM204V突变、25例(25/229,10.92%)rtM204I突变和2例(2/229,0.87%)rtM204V/I混合突变。下一代测序法检测到3例Sanger测序法漏检的rtM204V/I混合突变。与Sanger测序法相比,下一代测序法的灵敏度、特异度和准确度均为100%,突变类别完全符合率为95.95%(71/74),部分符合率为4.05%(3/74)。2种方法的一致性(κ)为0.97(P<0.01)。经重复性试验检测,下一代测序法的检测结果均一致,重复性符合率为100%。结论本研究建立的乙型肝炎病毒YMDD突变的下一代测序法灵敏度高,特异性好,重复性好,准确性高,比Sanger测序法更灵敏,能检测到更多的混合突变,为乙型肝炎病毒耐药基因检测提供了新的手段。更多还原

【Abstract】 Objective A next-generation sequencing(NGS) was established for the detection of hepatitis B virus(HBV)YMDD mutations and its performance was also evaluated. Methods DNA was extracted from serum samples by centrifugation column,and primers were designed to amplify the P gene region of HBV and the PCR products were captured by the target followed by NGS. The detection results were analyzed by using the bioinformatics software system to establish the NGS for the detection of HBV YMDD mutations. A total of 229 serum samples from patients with chronic HBV were collected. The NGS and Sanger sequencing were used to detect the YMDD gene mutations simultaneously,and the detection performance for NGS was evaluated. Five positive samples(including different types of gene mutations)and two negative samples were selected to detect repeatedly by NGS and the results were evaluated. Results Among 229 clinical samples,74(74/229,32.31%) YMDD mutations were detected by two sequencing methods. Of which 44(44/229,19.21%) were detected by NGS,25(25/229,10.92%) were detected by rtm204 i mutations,and 5(5/229,2.18%)rtm204 v/I mixed mutations. 47(47/229,20.52%)rtm204 v mutations,25(25/229,10.92%)rtm204 i mutations and 2(2/229,0.87%)rtm204 v/I mixed mutations were detected by Sanger sequencing. Three cases of rtm204 v/I mixed mutation missed by Sanger sequencing were detected by NGS. Compared with Sanger sequencing,the sensitivity,specificity and accuracy of NGS are100%. The complete coincidence rate was 95.95%(71/74),and the partial coincidence rate was 4.05%(3/74). The consistency of the two methods was 0.97(P<0.01). The repeatability coincidence rate between two methods is100%. Conclusion The NGS for the detection of HBV YMDD mutations has been established. This method is more sensitive than Sanger sequencing and can detect more mixed mutations providing a new method for the detection of drug-resistant genes of HBV.更多还原