Girón-Callejas Amalia, Clark Gemma, Irving William L, McClure C Patrick
School of Life Sciences, University of Nottingham, Nottingham, UK.
Department of Clinical Microbiology, Nottingham University Hospitals NHS Trust, Nottingham, UK.
J Virol Methods. 2015 Mar;214:25-8. doi: 10.1016/j.jviromet.2014.11.025. Epub 2014 Dec 18.
Hepatitis E virus (HEV) infection is a public health concern worldwide, associated with waterborne outbreaks in developing countries and reported as an emerging zoonotic infection in high-income countries. A recent consensus proposal classified the isolates from human, swine, wild boar, deer, mongoose, rabbit and camel in seven genotypes within the species Orthohepevirus A. In this report a popular HEV RT-qPCR assay was assessed for the detection of the species Orthohepevirus A. In silico analysis of 189 complete genome sequences showed that the assay targets a highly conserved region in the Orthohepevirus A genome. Additionally, plasmid standards were constructed to test the effect of probe- and primer-binding site mutations in the assay performance. The assay proved robust enough to detect strains with mutations in the probe-binding site and in the 3' end primer-binding site regions. A degenerate version of the reverse primer improves the performance of the assay particularly in the detection of HEV-5 and 6. The addition and detection of MS2 RNA in each RT-qPCR reaction monitored for amplification inhibition and did not affect the performance of the assay in the detection of the HEV RNA international standard. Therefore, the RT-qPCR assay can be confidently used for the RNA detection of the seven genotypes within the species Orthohepevirus A.
戊型肝炎病毒（HEV）感染是一个全球性的公共卫生问题，在发展中国家与水源性疫情暴发相关，在高收入国家则被报告为一种新出现的人畜共患感染。最近的一项共识提议将来自人类、猪、野猪、鹿、獴、兔和骆驼的分离株归为正戊型肝炎病毒属A种内的7个基因型。在本报告中，对一种常用的HEV RT-qPCR检测方法进行了评估，以检测正戊型肝炎病毒属A种。对189个完整基因组序列的电子分析表明，该检测方法针对的是正戊型肝炎病毒属A基因组中的一个高度保守区域。此外，构建了质粒标准品，以测试探针和引物结合位点突变对检测性能的影响。该检测方法被证明具有足够的稳健性，能够检测在探针结合位点和3'端引物结合位点区域发生突变的毒株。反向引物的简并版本提高了检测方法的性能，特别是在检测HEV-5和6时。在每个RT-qPCR反应中添加并检测MS2 RNA，以监测扩增抑制情况，且其不会影响该检测方法对HEV RNA国际标准品的检测性能。因此，RT-qPCR检测方法可可靠地用于正戊型肝炎病毒属A种内7个基因型的RNA检测。
