摘要目的 评价焦磷酸测序试剂盒(HBV DRT)检测HBV耐药的性能及临床适用性.方法 使用HBV核酸定量标准品梯度稀释系列样品验证试剂盒PCR检测下限;使用含HBV基因序列rt区片段、10个耐药相关突变位点均为野生型或均为突变型的2个质粒样品制备不同突变型/野生型比例的系列样品,重复检测至少10次用于评价试剂盒的准确性及建立突变比例真实值与检测值的线性关系;选取102例经Sanger测序分析rt区序列的核苷(酸)类药物治疗慢性乙型肝炎患者临床样本,使用HBV DRT试剂盒检测分析10个HBV耐药相关突变位点的特征,评价两种测序分析结果的一致性.结果 HBV DRT的PCR检测下限为50 IU/ml;线性分析提示除rt236位点外,其他9个耐药位点的突变比例真实值与检测值线性关系均达到R2＞ 0.98(P＜0.001);临床样本检测结果显示,4例样本部分位点PCR扩增失败,98例样本HBV DRT与Sanger测序检测结果比较,差异有统计学意义(x2=71.2,P＜0.001),总符合率达92.6％ (897/969),其中10个耐药相关突变位点结果全部一致的样本占46.9％(46/98).两种测序方法在10个位点上的一致率介于71.5％～ 100％;两种测序结果不一致中,87.5％ (63/72)为Sanger测序结果为野生型(WT)而HBV DRT检测结果为WT/突变混合型(MT),6.9％ (5/72)为Sanger测序结果为WT而HBV DRT检测结果为MT,5.6％ (4/72)为Sanger测序结果为WT/MT而HBVDRT检测结果为WT.结论 HBV DRT试剂盒检测乙型肝炎耐药基因具有良好的敏感性和准确性,在检测样本中存在少量病毒突变株时优于直接测序,可用于耐药突变的早期检测.

abstractsObjective To evaluate the quality and clinical applicability ofpyrosequencing assay kit for detecting hepatitis B virus resistance (HBV DRT).Methods Serial dilutions of the International Standard for HBV DNA were used to test the detection limit of the PCR for HBV DRT.Plasmids containing the either a wild-type (WT) copy or one of 10 mutant (MT) copies of the HBV RT gene were used to prepare a series of samples with various mutation ratios.To construct the linear relationship between the true mutation rate and the detected mutation rate,each sample was repeated at least 10 times.A total of 102 clinical samples were analyzed by Sanger sequencing and retested by the PCR for HBV DRT to determine the concordance of these two methods.Results The lower detection limit of the PCR for HBV DRT was 50 IU/ml.Except for the RT236 MT,the correlation between the true mutation rate and the detected mutation rate for the other nine resistance-related mutation sites were excellent,with R2＞0.98 (P ＜ 0.001).Among the 102 clinical samples,four were not amplified successfully by PCR.The results were significantly different between the PCR for HBV DRT method and the Sanger sequencing method (x2 =71.2,P ＜ 0.001),and concordance was observed for 897/969 (92.6％) amino acid positions in 98 samples.Concordant results were achieved in 46/98 (46.9％) samples at all 10 mutation sites.For detection of a single mutation site,concordance rates ranged from 71.5％ to 100％ at the 10 mutation sites,respectively.Analysis of discordant samples showed that in 87.5％ (63/72),Sanger sequencing detected WT and the PCR for HBV DRT detected WT/MT.In 5.6％ (4/72) of samples,Sanger sequencing detected WT/MT and the PCR for HBV DRT detected WT.In the remaining 6.9％ (5/72) of samples,Sanger sequencing detected WT but PCR for HBV DRT detected MT.Conclusion The PCR for HBV DRT showed high sensitivity and accuracy in detecting antiviral drug-resistant mutations.The method is superior to Sanger sequencing for detecting minor mutations and can be used for early detection of a resistance mutation.