Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR
Detection of norovirus (GI, GII), Sapovirus and astrovirus in fecal samples using reverse transcription single-round multiplex PCR
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DOI：
10.1016/j.jviromet.2003.08.009
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发表时间：
2003-12-01
影响因子：
3.1
通讯作者：
Ushijima, H
中科院分区：
文献类型：
--
作者：
Yan, HN;Yagyu, F;Ushijima, H
开发了一种逆转录 (RT) 单轮多重聚合酶链反应 (smPCR) 检测方法，可同时检测诺如病毒基因组 I 和 H、沙波病毒和星状病毒。 2000 年 7 月至 2001 年 6 月期间，对来自日本四个地区的总共 377 份腹泻粪便样本（轮状病毒和腺病毒阴性筛查）进行了 RT-smPCR 检测。阳性率为16.4%（377份粪便样本中的62份）。 60 份阳性样本中，分别检测出 42 份、16 份和 4 份诺如病毒、沙波病毒和星状病毒。这些样本中未发现混合感染。感染主要发生在11月、12月和1月。 RT-smPCR 的关键要素是 (i) 使用 Superscript RTII 和随机引物在 42°C 下合成 1 小时，在 99°C 下 5 分钟，以及 (ii) 使用 Taq 聚合酶与四种不同引物对的混合物混合进行单轮多重 PCR（诺如病毒基因组 I 为 G1-SKF/G1-SKR，诺如病毒基因组为 COG2F/G2-SKR） 11、SLV5317/SLV5749（沙波病毒）、PreCAP1/82b（星状病毒）。所有四个引物对均扩增目标病毒基因组的衣壳区域，分别针对诺如病毒基因组 I 和 11、沙波病毒和星状病毒产生 330、387、434、719 bp 的四个大小特异性扩增子。该检测提供了一种更快速、更有效的方法，可以在一次测试中从粪便样本中检测这些病毒，并且还为食品和环境样本中的分子检测提供了潜力。 (C) 2003 Elsevier B.V. 保留所有权利。
A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and H, Sapovirus and astrovirus. A total of 377 diarrhea stool samples (screened for rotavirus- and adenorivus-negative) from four regions in Japan during July 2000 to June 2001 were examined by RT-smPCR. The positive rate was 16.4% (62 out of 377 stool samples). Norovirus, Sapovirus and astrovirus were detected in 42, 16, 4 of 60 positive samples, respectively. Coinfection was not found in these samples. Infections occurred mainly in November, December and January. The key elements of the RT-smPCR are (i) the cDNA synthesis with the Superscript RTII and random primer at 42degreesC for 1 h, at 99degreesC for 5 min, and (ii) single-round multiplex PCR by using Taq polymerase mixed together with a mixture of four different primer pairs (G1-SKF/G1-SKR for Norovirus genogroup I, COG2F/G2-SKR for Norovirus genogroup 11, SLV5317/SLV5749 for Sapovirus, PreCAP1/82b for astrovirus). All of the four primer pairs amplify the capsid region of target viral genome, produce four size-specific amplicons of 330, 387, 434, 719 bp for Norovirus genogroup I and 11, Sapovirus and astrovirus, respectively. This assay provides a more rapid and efficient way to detect these viruses from fecal samples in a single test, and also offers the potential for their molecular detection in food and environmental samples. (C) 2003 Elsevier B.V. All rights reserved.