Microbiology Department, Cliniques universitaires St Luc, Université catholique de Louvain, Brussels, Belgium.
Pôle de Microbiologie médicale, Institut de Recherche Expérimentale et Clinique, Université catholique de Louvain , Brussels, Belgium.
BMC Infect Dis. 2019 Aug 22;19(1):738. doi: 10.1186/s12879-019-4386-4.
The technique most frequently used to genotype HCV is quantitative RT-PCR. This technique is unable to provide an accurate genotype/subtype for many samples; we decided to develop an in-house method with the goal of accurately identifying the genotype of all samples. As a Belgium National Centre of reference for hepatitis, we developed in-house sequencing not only for 5'UTR and core regions starting from VERSANT LiPA amplicons but also for NS5B regions. The sequencing of VERSANT LiPA amplicons might be useful for many laboratories worldwide using the VERSANT LiPA assay to overcome undetermined results.
100 samples from Hepatitis C virus infected patients analysed by the VERSANT HCV Genotype 2.0 LiPA Assay covering frequent HCV types and subtypes were included in this study. NS5B, 5'UTR and Core home-made sequencing were then performed on these samples. The sequences obtained were compared with the HCV genomic BLAST bank.
All the samples were characterised by the VERSANT LiPA assay (8 G1a, 17 G1b, 6 G2, 11 G3, 13 G4, and 10 G6). It was not possible to discriminate between G6 and G1 by the VERSANT LiPA assay for 8 samples and 27 had an undetermined genotype. Forty-one samples were sequenced for the three regions: NS5B, 5'UTR and Core. Twenty-three samples were sequenced for two regions: 5' UTR and Core and 36 samples were sequenced only for NS5B. Of the 100 samples included, 64 samples were analysed for 5'UTR and Core sequencing and 79 samples were analysed for NS5B sequencing. The global agreement between VERSANT LiPA assay and sequencing was greater than 95%.
In this study, we describe a new, original method to confirm HCV genotypes of samples not discriminated by a commercial assay, using amplicons already obtained by the screening method, here the VERSANT LiPA assay. This method thus saves one step if a confirmation assay is needed and might be of usefulness for many laboratories worldwide performing VERSANT LiPA assay in particular.
目前最常用于 HCV 基因分型的技术是定量 RT-PCR。对于许多样本，该技术无法提供准确的基因型/亚型；因此，我们决定开发一种内部方法，以准确识别所有样本的基因型。作为比利时肝炎国家参考中心，我们不仅针对 VERSANT LiPA 扩增子的 5'UTR 和核心区域，还针对 NS5B 区域开发了内部测序。对于全球许多使用 VERSANT LiPA 检测方法的实验室来说，测序 VERSANT LiPA 扩增子可能有助于克服不确定的结果。
这项研究纳入了 100 例经 VERSANT HCV Genotype 2.0 LiPA 分析的丙型肝炎病毒感染患者样本，这些样本涵盖了常见的 HCV 类型和亚型。然后对这些样本进行 NS5B、5'UTR 和核心区域的内部测序。所获得的序列与 HCV 基因组 BLAST 数据库进行比较。
所有样本均通过 VERSANT LiPA 检测（8 例 G1a、17 例 G1b、6 例 G2、11 例 G3、13 例 G4 和 10 例 G6）。8 例 G6 和 G1 样本无法通过 VERSANT LiPA 检测区分，27 例样本基因型不确定。对 41 例样本的三个区域（NS5B、5'UTR 和核心）进行了测序。对 23 例样本的 5'UTR 和核心区域进行了测序，对 36 例样本仅对 NS5B 区域进行了测序。在纳入的 100 例样本中，64 例样本进行了 5'UTR 和核心测序分析，79 例样本进行了 NS5B 测序分析。 VERSANT LiPA 检测与测序之间的总体一致性大于 95%。
在这项研究中，我们描述了一种新的、原创的方法，用于确认通过商业检测方法无法区分的 HCV 样本的基因型，使用的是筛选方法（这里是 VERSANT LiPA 检测）已经获得的扩增子。因此，如果需要确认检测，该方法可以节省一个步骤，对于全球许多进行 VERSANT LiPA 检测的实验室尤其有用。
