Walker Andreas, Bergmann Matthias, Camdereli Jennifer, Kaiser Rolf, Lübke Nadine, Timm Jörg
Institute of Virology, Heinrich-Heine-University, University Hospital, Düsseldorf, Germany.
Institute of Virology, University of Cologne, Cologne, Germany.
J Clin Virol. 2017 Jun;91:42-48. doi: 10.1016/j.jcv.2017.04.008. Epub 2017 Apr 10.
HCV treatment options and cure rates have tremendously increased in the last decade. Although a pan-genotype HCV treatment has recently been approved, most DAA therapies are still genotype specific. Resistance-associated variants (RAVs) can limit the efficacy of DAA therapy and are associated with increased risk for therapy failure. With the approval of DAA regimens that recommend resistance testing prior to therapy, correct assessment of the genotype and testing for viruses with RAVs is clinically relevant. However, genotyping and resistance testing is generally done in costly and laborious separate reactions.
The aim of the study was to establish a genotype-independent full-genome reverse transcription protocol to generate a template for both genotyping and resistance testing and to implement it into our routine diagnostic setup.
The complete HCV genome was reverse transcribed with a pan-genotype primer binding at the 3'end of the viral RNA. This cDNA served as template for transcription of the genotyping amplicon in the core region as well as for the resistance testing of NS3, NS5A, and NS5B.
With the established RT-protocol the HCV core region was successfully amplified and genotyped from 124 out of 125 (99.2%) HCV-positive samples. The amplification efficiency of RAV containing regions in NS3, NS5A, NS5B was 96.2%, 96.6% and 94.4%, respectively.
We developed a method for HCV full-genome cDNA synthesis and implemented it into a routine diagnostic setup. This cDNA can be used as template for genotyping amplicons covering the core or NS5B region as well as for resistance testing amplicons in NS3, NS5A and NS5B.
在过去十年中，丙型肝炎病毒（HCV）的治疗选择和治愈率大幅提高。尽管最近一种泛基因型HCV治疗方法已获批准，但大多数直接抗病毒药物（DAA）疗法仍具有基因型特异性。耐药相关变异体（RAV）会限制DAA疗法的疗效，并与治疗失败风险增加相关。随着推荐在治疗前进行耐药检测的DAA方案获得批准，准确评估基因型以及检测带有RAV的病毒在临床上具有重要意义。然而，基因分型和耐药检测通常在成本高昂且繁琐的单独反应中进行。
本研究的目的是建立一种不依赖基因型的全基因组逆转录方案，以生成用于基因分型和耐药检测的模板，并将其应用于我们的常规诊断流程。
使用一种在病毒RNA 3'端结合的泛基因型引物对完整的HCV基因组进行逆转录。该cDNA用作核心区域基因分型扩增子转录的模板，以及NS3、NS5A和NS5B耐药检测的模板。
采用已建立的逆转录方案，125份HCV阳性样本中有124份（99.2%）成功扩增并对HCV核心区域进行了基因分型。NS3、NS5A、NS5B中含有RAV区域的扩增效率分别为96.2%、96.6%和94.4%。
我们开发了一种HCV全基因组cDNA合成方法，并将其应用于常规诊断流程。该cDNA可作为覆盖核心或NS5B区域的基因分型扩增子以及NS3、NS5A和NS5B耐药检测扩增子的模板。
