Division of Infection and Immunity, University College London, London, UK.
J Virol Methods. 2012 Dec;186(1-2):157-60. doi: 10.1016/j.jviromet.2012.07.027. Epub 2012 Jul 31.
Hepatitis E virus (HEV) is a major cause of acute viral hepatitis in many parts of the developing world. It is responsible for both sporadic infections and large scale epidemics and may be associated with significant mortality during pregnancy. Over the past two decades many serological and nucleic acid based diagnostic tests for HEV have been developed, including several reverse transcription real-time polymerase chain reaction assays (RT-qPCR). One of the most widely used of these RT-qPCRs is that developed by Jothikumar and colleagues (Journal of Virological Methods 2006, 131, 65-71). Whilst reviewing this assay we calculated the predicted melting temperature of its TaqMan probe and consequently synthesised a minor groove binder (MGB) version in order to increase its hybridisation stability. In this report the performance of the original unmodified probe is compared with that of the MGB-modified version. We demonstrate that the MGB-modified probe detected HEV RNA in plasma samples from six patients with serologically confirmed hepatitis E in whom the unmodified probe had failed to detect HEV RNA. Sequence analysis of the ORF3 segment targeted by the RT-qPCR was possible in 4 of the 6 patients and revealed an identical C→T single nucleotide mutation in the probe binding region in each case.
戊型肝炎病毒 (HEV) 是发展中国家许多地区急性病毒性肝炎的主要病因。它既能引起散发性感染，也能引起大规模流行，而且在妊娠期间可能导致严重的死亡率。在过去的二十年中，已经开发出了许多针对 HEV 的血清学和基于核酸的诊断检测方法，包括几种逆转录实时聚合酶链反应检测（RT-qPCR）。其中最广泛使用的 RT-qPCR 之一是 Jothikumar 及其同事开发的（Journal of Virological Methods 2006, 131, 65-71）。在对该检测方法进行评估时，我们计算了 TaqMan 探针的预测熔解温度，并随后合成了一个小沟结合物（MGB）版本，以提高其杂交稳定性。在本报告中，比较了原始未修饰探针与 MGB 修饰版本的性能。我们证明，在 6 名经血清学确认患有戊型肝炎的患者的血浆样本中，MGB 修饰探针能够检测到 HEV RNA，而未修饰探针未能检测到 HEV RNA。对 RT-qPCR 靶向的 ORF3 片段进行序列分析，在 4 名患者中进行了分析，发现每个患者的探针结合区域都存在相同的 C→T 单核苷酸突变。
