Yan Hainian, Yagyu Fumihiro, Okitsu Shoko, Nishio Osamu, Ushijima Hiroshi
Department of Developmental Medical Sciences, Graduate School of Medicine, Institute of International Health, The University of Tokyo, 7-3-1 Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
J Virol Methods. 2003 Dec;114(1):37-44. doi: 10.1016/j.jviromet.2003.08.009.
A reverse transcription (RT) single-round multiplex polymerase chain reaction (smPCR) assay was developed to detect simultaneously Norovirus genogroup I and II, 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 42 degrees C for 1 h, at 99 degrees C 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 II, 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 II, 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.
开发了一种逆转录（RT）单轮多重聚合酶链反应（smPCR）检测方法，用于同时检测诺如病毒I基因组和II基因组、札如病毒和星状病毒。对2000年7月至2001年6月期间从日本四个地区采集的377份腹泻粪便样本（经检测轮状病毒和腺病毒均为阴性）进行了RT-smPCR检测。阳性率为16.4%（377份粪便样本中有62份）。在60份阳性样本中，分别检测到42份诺如病毒、16份札如病毒和4份星状病毒。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的关键步骤包括：（i）使用Superscript RTII和随机引物在42℃下合成cDNA 1小时，然后在99℃下保温5分钟；（ii）使用Taq聚合酶与四种不同引物对的混合物（诺如病毒I基因组用G1-SKF/G1-SKR，诺如病毒II基因组用COG2F/G2-SKR，札如病毒用SLV5317/SLV5749，星状病毒用PreCAP1/82b）进行单轮多重PCR。所有这四对引物均扩增目标病毒基因组的衣壳区域，分别产生诺如病毒I基因组和II基因组、札如病毒和星状病毒的四种大小特异性扩增子，大小分别为330、387、434、719 bp。该检测方法提供了一种更快速、高效的方法，可在单次检测中从粪便样本中检测这些病毒，同时也为在食品和环境样本中进行分子检测提供了可能。
