Oka Tomoichiro, Katayama Kazuhiko, Hansman Grant S, Kageyama Tsutomu, Ogawa Satoko, Wu Fang-Tzy, White Peter A, Takeda Naokazu
Department of Virology II, National Institute of Infectious Diseases, Musashi-Murayama, Tokyo, Japan.
J Med Virol. 2006 Oct;78(10):1347-53. doi: 10.1002/jmv.20699.
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.
札幌病毒（SaV）是一种引起人类和猪肠胃炎的病原体，根据其衣壳基因序列可分为五个不同的基因群（GI - GV）。检测SaV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）。一种基于TaqMan的新型实时RT-PCR检测方法被开发出来，它灵敏度高，能够检测多种基因多样的人类SaV毒株。对10个全长SaV基因组序列进行核苷酸比对并进行相似性图谱分析，结果表明最保守的位点是开放阅读框1（ORF1）中的聚合酶 - 衣壳连接区。基于对编码该连接区的27个可用序列的多重比对，我们设计了引物组和TaqMan MGB探针，能够在单个管中检测人类SaV GI、GII、GIV和GV序列。用SaV GI、GII、GIV和GV对照质粒证实了其反应性，每管的检测效率范围为2.5×10⁷至2.5×10¹拷贝。使用临床粪便标本进行分析表明，该系统能够检测SaV GI、GII、GIV和GV序列，并且未观察到与其他肠道病毒（包括诺如病毒（NoV）、轮状病毒、星状病毒和腺病毒）的交叉反应。这是首个能够检测人类札幌病毒所有基因群的实时RT-PCR系统。
