巢式PCR联合焦磷酸测序法在乙型肝炎病毒耐药基因检测中的敏感性与特异性分析

Sensitivity and specificity of nested PCR pyrosequencing in hepatitis B virus drug resistance gene testing

目的评价巢式PCR联合焦磷酸测序法在乙型肝炎病毒(HBV)耐药基因检测中的敏感与特异性。方法选取rtM204I(ATT)突变型和rtM204(ATG)未突变型质粒,按照不同比例混合后,采用巢式PCR联合焦磷酸测序法对突变位点进行检测,并与常规PCR焦磷酸测序法进行比较,对两种方法的线性及一致性进行分析;选取不同病毒载量临床标本,采用巢式PCR焦磷酸测序法检测耐药突变位点,并与巢式PCR Sanger测序法进行比较,对两种方法的敏感性及特异性进行分析。结果常规PCR焦磷酸测序和巢式PCR焦磷酸测序结果拟合曲线R2>0.99,P<0.05,线性模型有显著性意义。分别将常规PCR焦磷酸测序和巢式PCR焦磷酸测序结果与预测值的差值进行分析,巢式PCR能够较常规PCR更好的反映实际值,在90%比例处优于常规PCR。临床75例患者标本巢式PCR Sanger测序与巢式PCR联合焦磷酸测序结果灵敏性比较提示,Sanger测序的灵敏性为92%,巢式PCR联合焦磷酸测序的灵敏性达到100%,差异有统计学意义(P<0.01)。对于不同病毒拷贝数标本的灵敏性不同,其差异主要表现在低浓度组(HBV DNA≤3log10 copies/ml),Sanger测序的灵敏性为78%,而焦磷酸测序的灵敏性可以达到100%,差异有统计学意义(P<0.01)。阴性对照,两种方法均未检出HBV病毒,两种方法的特异性均较好。结论巢式PCR联合焦磷酸测序的方法监测乙肝病毒变异同巢式PCR联合sanger测序相比,有更好的灵敏性,尤其是在低病毒拷贝数时,差异明显,这对于临床提早发现耐药突变株,指导临床及时更换用药有重要意义。

Objective To evaluate the sensitivity and specificity of nested PCR combined with pyrosequencing in the detection of HBV drug-resistance gene. Methods RtM204I （ATT） mutant and rtM204 （ATG） nonmutant plasmids mixed at different ratios were detected for mutations using nested-PCR combined with pyrosequencing, and the results were compared with those by conventional PCR pyrosequencing to analyze the linearity and consistency of the two methods. Clinical specimens with different viral loads were examined for drug-resistant mutations using nested PCR pyrosequencing and nested PCR combined with dideoxy sequencing （Sanger） for comparison of the detection sensitivity and specificity. Results The fitting curves demonstrated good linearity of both conventional PCR pyrosequencing and nested PCR pyrosequencing （R2〉0.99, P〈 0.05）. Nested PCR showed a better consistency with the predicted value than conventional PCR, and was superior to conventional PCR for detection of samples containing 90% mutant plasmid. In the detection of clinical specimens, Sanger sequencing had a significantly lower sensitivity than nested PCR pyrosequencing （92% vs 100%, P〈0.01）. The detection sensitivity of Sanger sequencing varied with the viral loads, especially in samples with low viral copies （HBV DNA ~31og10 copies/ml）, where the sensitivity was 78%, significantly lower than that of pyrosequencing （100%, P〈0.01）. Neither of the two methods yielded positive results for the negative control samples, suggesting their good specificity. Conclusion Compared with nested PCR and Sanger sequencing method, nested PCR pyrosequencing has a higher sensitivity especially in clinical specimens with low viral copies, which can be important for early detection of HBV mutant strains and hence more effective clinical management.