A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus
A broadly reactive one-step real-time RT-PCR assay for rapid and sensitive detection of hepatitis E virus
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DOI：
10.1016/j.jviromet.2005.07.004
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发表时间：
2006-01-01
影响因子：
3.1
通讯作者：
Hill, VR
中科院分区：
文献类型：
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作者：
Jothikumar, N;Cromeans, TL;Hill, VR
戊型肝炎病毒（HEV）通过粪-口途径传播，引起散发性和流行性急性肝炎。据记载，大规模的水媒HEV流行仅发生在发展中国家。全世界至少报告了四种主要的HEV基因型：基因型I（主要在亚洲国家发现），基因型2（从墨西哥的单一爆发中分离），基因型3（在美国和许多其他国家的猪和人类中鉴定）和基因型4（在亚洲的人类，猪和其他动物中鉴定）。为了更好地检测和定量临床和环境样本中可能存在的不同戊型肝炎病毒株，我们开发了一种快速灵敏的实时RT-PCR检测方法来检测戊型肝炎病毒RNA。根据ORF 3区27个序列的多重序列比对，选择用于实时RT-PCR的引物和探针。代表基因型1-4的13个HEV分离株用于标准化实时RT-PCR测定。TaqMan（R）测定检测到少至4个基因组当量（GE）拷贝的HEV质粒DNA，并且检测到低至0.12 50%猪感染剂量（PID 50）的猪HEV。在实时RT-PCR试验中检测了掺入地表水浓缩物中的不同浓度的猪HEV（120-1.2 PID 50）。这是第一次报道广泛反应性TaqMan（R）RT-PCR检测临床和环境样本中的HEV。(c)2005 Elsevier B. V.保留所有权利。
Hepatitis E virus (HEV) is transmitted by the fecal-oral route and causes sporadic and epidemic forms of acute hepatitis. Large waterborne HEV epidemics have been documented exclusively in developing countries. At least four major genotypes of HEV have been reported worldwide: genotype I (found primarily in Asian countries), genotype 2 (isolated from a single outbreak in Mexico), genotype 3 (identified in swine and humans in the United States and many other countries), and genotype 4 (identified in humans, swine and other animals in Asia). To better detect and quantitate different HEV strains that may be present in clinical and environmental samples, we developed a rapid and sensitive real-time RT-PCR assay for the detection of HEV RNA. Primers and probes for the real-time RT-PCR were selected based on the multiple sequence alignments of 27 sequences of the ORF3 region. Thirteen HEV isolates representing genotypes 1-4 were used to standardize the real-time RT-PCR assay. The TaqMan (R) assay detected as few as four genome equivalent (GE) copies of HEV plasmid DNA and detected as low as 0.12 50% pig infectious dose (PID50) Of swine HEV. Different concentrations of swine HEV (120-1.2 PID50) spiked into a Surface water concentrate were detected in the real-time RT-PCR assay. This is the first reporting of a broadly reactive TaqMan (R) RT-PCR assay for the detection of HEV in clinical and environmental samples. (c) 2005 Elsevier B.V. All rights reserved.