Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans
Application of a reverse transcription-PCR for identification and differentiation of Aichi virus, a new member of the picornavirus family associated with gastroenteritis in humans
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DOI：
10.1128/jcm.38.8.2955-2961.2000
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发表时间：
2000-08-01
影响因子：
9.4
通讯作者：
Miyazaki, Y
中科院分区：
文献类型：
--
作者：
Yamashita, T;Sugiyama, M;Miyazaki, Y
从日本5次胃肠炎爆发的7名患者、5名从东南亚国家返回的日本人和5名患有胃肠炎的巴基斯坦当地儿童中分离的Vero细胞中的爱知病毒，根据其与标准株（A846/88）的单克隆抗体的反应性和3个基因组区域的逆转录-PCR（RT-PCR），进行了鉴别检查。在3C的C端和3D的N端之间的假定连接处，确定了这17个分离物的519个碱基的RNA序列。分析显示这些分离物之间的同源性约为90%，然后将其分为两组：组1（基因型A）包括来自4次暴发的6株分离株和来自旅行者和组2的1株分离株（基因型B）包括来自另一次爆发的一个分离株、来自回国旅行者的四个分离株以及来自巴基斯坦儿童的所有分离株。根据分离株序列设计引物和生物素标记探针，对粪便标本中AichiRNA的3C-3D连接处223个碱基进行扩增和检测。在日本37起胃肠炎暴发疫情的12例（32%）患者的99份粪便标本中，有54份（55%）检出AichiRNA。在12起疫情中，11起疑似由A基因型引起。这些结果表明，RT-PCR可以是一个有用的工具来检测粪便样本中的爱知病毒，PCR产物的序列分析，可以用来确定在每个事件中的流行株。
Aichi viruses isolated in Vero cells from seven patients in five gastroenteritis outbreaks in Japan, five Japanese returning from Southeast Asian countries, and five local children in Pakistan with gastroenteritis were examined for differentiation based on their reactivities with a monoclonal antibody to a standard strain (A846/88) and a reverse transcription-PCR (RT-PCR) of three genomic regions. The RNA sequences were determined for 519 bases of these 17 isolates at the putative junction between the C terminus of 3C and the N terminus of 3D, The analyses revealed an approximately 90% homology between these isolates, which were then divided into two groups: group 1 (genotype A) included six isolates from four outbreaks and one isolate from a traveler and group 2 (genotype B) included one isolate from the other outbreak, four isolates from returning travelers, and all of the isolates from the Pakistani children. Based on the isolate sequences, a primer pair and a biotin-labeled probe were designed for amplification and detection of 223 bases at the 3C-3D junction of Aichi virus RNA in fecal specimens, The Aichi virus RNA was detected in 54 (55%) of 99 fecal specimens from the patients in 12 (32%) of 37 outbreaks of gastroenteritis in Japan. Of the 12 outbreaks, 11 were suspected to be due to genotype A. These results indicated that RT-PCR can be a useful tool to detect Aichi virus in stool samples and that a sequence analysis of PCR products can be employed to identify the prevalent strain in each incident.