Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays
Development of a TaqMan assay for the six major genotypes of hepatitis C virus: Comparison with commercial assays
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DOI：
10.1002/jmv.21043
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发表时间：
2008-01-01
影响因子：
12.7
通讯作者：
Bukh, Jens
中科院分区：
文献类型：
--
作者：
Engle, Ronald E.;Russell, Rodney S.;Bukh, Jens
建立了一种实时荧光定量PCR检测方法，用于检测代表6种主要基因型丙型肝炎病毒（HCV）的参考菌株的基因组RNA，具有相同的灵敏度，并能准确测量JFH1型HCV感染的Huh7.5细胞中的HCV RNA。该方法间接校准为首个国际（WHO 96/790） HCV标准制剂，线性动态范围为10(2.6)~ 10(6.5)lU/Ml。此外，测定间和测定内的精密度分别接近3% CV和< 2% CV。与同样采用世卫组织标准的市售HCV RNA核酸技术试剂盒（Versant HCV RNA 3.0 b-DNA和Amplicor HCV Monitor）获得的结果进行比较，验证了TaqMan检测对所有主要HCV基因型的有效性。两种商业方法在很宽的动态范围内检测HCV RNA，但在评估不同HCV基因型样本时显示出约0.3 log（10）的一致性差异。三种方法获得的基因组滴度与先前确定的HCV参考株的感染性滴度相关。TaqMan检测已成为临床样本和细胞培养实验中跟踪病毒载量的重要工具，该技术在线性动态范围、灵敏度和定制方面具有显著优势。
A quantitative real-time PCR assay was developed that detects genomic RNA from reference strains representing the six major genotypes of hepatitis C virus (HCV) with equal sensitivity and accurately measured HCV RNA in JFH1 HCV-infected Huh7.5 cells. The method is indirectly calibrated to the first international (WHO 96/790) HCV standard preparation and has a linear dynamic range of 10(2.6)-10(6.5) lU/Ml. In addition, the inter- and intra-assay precision were similar to 3% CV and < 2% CV, respectively. Comparison with results obtained by commercially available HCV RNA Nucleic Acid Technology kits (Versant HCV RNA 3.0 b-DNA and Amplicor HCV Monitor), that also employ the WHO standard, allowed validation of the TaqMan assay against all major HCV genotypes. Both commercial methods detected HCV RNA over a wide dynamic range, but showed a consistent difference of about 0.3 log(10) when evaluating samples of different HCV genotypes. The genome titers obtained with the three methods correlated with the infectivity titers previously determined for the HCV reference strains. TaqMan assays have become an essential tool to follow viral load in clinical samples and cell culture-based experiments and this technology offers significant advantages in linear dynamic range, sensitivity and customization.