Lowe Christopher F, Merrick Linda, Harrigan P Richard, Mazzulli Tony, Sherlock Christopher H, Ritchie Gordon
Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada Department of Pathology and Laboratory Medicine, University of British Columbia, Vancouver, British Columbia, Canada
Division of Microbiology and Virology, Providence Health Care, Vancouver, British Columbia, Canada.
J Clin Microbiol. 2016 Jan;54(1):127-33. doi: 10.1128/JCM.02229-15. Epub 2015 Nov 4.
Sanger sequencing or DNA hybridization have been the primary modalities for hepatitis B (HBV) resistance testing and genotyping; however, there are limitations, such as low sensitivity and the inability to detect novel mutations. Next-generation sequencing (NGS) for HBV can overcome these limitations, but there is limited guidance for clinical microbiology laboratories to validate this novel technology. In this study, we describe an approach to implementing deep pyrosequencing for HBV resistance testing and genotyping in a clinical virology laboratory. A nested PCR targeting the pol region of HBV (codons 143 to 281) was developed, and the PCR product was sequenced by the 454 Junior (Roche). Interpretation was performed by ABL TherapyEdge based on European Association for the Study of the Liver (EASL) guidelines. Previously characterized HBV samples by INNO-LiPA (LiPA) were compared to NGS with discordant results arbitrated by Sanger sequencing. Genotyping of 105 distinct samples revealed a concordance of 95.2% (100/105), with Sanger sequencing confirming the NGS result. Resistance testing by NGS was concordant with LiPA in 85% (68/80) of previously characterized samples. Additional mutations were found in 8 samples, which related to the identification of low-level mutant subpopulations present at <10% (6/8). To balance the costs of testing for the validation study, reproducibility of the NGS was investigated through an analysis of sequence variants at loci not associated with resistance in a single patient sample. Our validation approach attempts to balance costs with efficient data acquisition.
桑格测序法或DNA杂交一直是乙肝病毒（HBV）耐药性检测和基因分型的主要方法；然而，这些方法存在局限性，如灵敏度低以及无法检测新突变。用于HBV的新一代测序（NGS）技术可以克服这些局限性，但临床微生物实验室在验证这项新技术方面的指导有限。在本研究中，我们描述了一种在临床病毒学实验室中实施焦磷酸测序法进行HBV耐药性检测和基因分型的方法。我们开发了一种针对HBV pol区（密码子143至281）的巢式PCR，PCR产物通过454 Junior（罗氏公司）进行测序。由ABL TherapyEdge根据欧洲肝脏研究协会（EASL）指南进行解读。将先前通过INNO-LiPA（LiPA）法鉴定的HBV样本与NGS结果进行比较，不一致的结果通过桑格测序法进行仲裁。对105个不同样本进行基因分型，结果显示一致性为95.2%（100/105），桑格测序法证实了NGS结果。在80个先前已鉴定的样本中，NGS耐药性检测结果与LiPA法的一致性为85%（68/80）。在8个样本中发现了其他突变，这与鉴定出存在比例<10%的低水平突变亚群有关（6/8）。为了平衡验证研究的检测成本，通过分析单个患者样本中与耐药性无关位点的序列变异来研究NGS的可重复性。我们的验证方法试图在成本与高效数据采集之间取得平衡。
