Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction
Detection of human sapovirus by real-time reverse transcription-polymerase chain reaction
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DOI：
10.1002/jmv.20699
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发表时间：
2006-10-01
影响因子：
12.7
通讯作者：
Takeda, Naokazu
中科院分区：
文献类型：
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作者：
Oka, Tomoichiro;Katayama, Kazuhiko;Takeda, Naokazu
Sapovirus（SaV）是一种引起人和猪胃肠炎的病毒，根据其衣壳蛋白基因序列可分为5个不同的基因组（GI-GV）。SaV检测的典型方法包括电子显微镜（EM）、酶联免疫吸附测定（ELISA）和逆转录聚合酶链反应（RT-PCR）。开发了一种新的基于TaqMan的实时RT-PCR检测方法，该方法灵敏度高，能够检测广泛的遗传多样性人SaV毒株。对10个SaV全基因组序列进行相似性分析，发现开放阅读框1（ORF 1）中的聚合酶-衣壳连接位点最为保守。基于编码该连接的27个可用序列的多重比对，我们设计了一组引物和TaqMan MGB探针，用于在单管中检测人SaV GI、GII、GIV和GV序列。用SaV GI、GII、GIV和GV对照质粒证实了反应性，效率范围为2.5 x 10（7）至2.5 x 10（1）拷贝/管。使用临床粪便标本的分析显示，本系统能够检测SaV GI、GII、GIV和GV序列，并且没有观察到对其他肠道病毒的交叉反应性，包括诺如病毒（NoV）、轮状病毒、星状病毒和腺病毒。这是第一个可以检测所有基因组的人痘病毒的实时RT-PCR系统。
Sapovirus (SaV) is an agent of gastroenteritis for humans and swine, and is divided into five distinct genogroups (GI-GV) based on its capsid gene sequences. Typical methods of SaV detection include electron microscopy (EM), enzyme-linked immunosorbent assay (ELISA), and reverse transcription-polymerase chain reaction (RT-PCR). A novel TaqMan-based real-time RT-PCR assay was developed that is sensitive and has the ability to detect the broad range of genetically diverse human SaV strains. A nucleotide alignment of 10 full-length SaV genome sequences was subjected to similarity plot analysis, which indicated that the most conserved site was the polymerase-capsid junction in open reading frame 1 (ORF1). Based on multiple alignments of the 27 available sequences encoding this junction, we designed sets of primers and TaqMan MGB probes that detect human SaV GI, GII, GIV, and GV sequences in a single tube. The reactivity was confirmed with SaV GI, GII, GIV, and GV control plasmids, and the efficiency ranged from 2.5 x 10(7) to 2.5 x 10(1) copies per tube. Analysis using clinical stool specimens revealed that the present system was capable of detecting SaV GI, GII, GIV, and GV sequences, and no cross-reactivity was observed against other enteric viruses, including norovirus (NoV), rotavirus, astrovirus, and adenovirus. This is the first real-time RT-PCR system that could detect all genogroups of human sapoviruses.