Ishida Setsuko, Yoshizumi Shima, Ikeda Tetsuya, Miyoshi Masahiro, Okano Motohiko, Okui Toyo
Division of Enteric Virology, Department of Microbiology, Hokkaido Institute of Public Health, Sapporo, Japan.
J Med Virol. 2008 May;80(5):913-20. doi: 10.1002/jmv.21142.
Conventional reverse transcription-polymerase chain reaction (RT-PCR) to detect norovirus (NV) is a complex of multi-step procedure that requires gel electrophoresis as well as hybridization or sequencing to confirm the final diagnosis. A duplex TaqMan RT-PCR was developed to detect and classify genogroup (G) I and GII of NV. The primers and TaqMan probes for this assay were selected from the region of open reading frame (ORF) 1-ORF2 junction. A total of 796 stool specimens from 103 outbreaks of gastroenteritis, and a series of 46 stool specimens containing most NV genotypes was used for this study. For these specimens from 103 outbreaks, NV was detected and classified by the duplex TaqMan RT-PCR in 536 of the 541 specimens tested previously to be positive using the conventional RT-PCR. Two hundred fifty-one of the 255 specimens that were negative by the conventional RT-PCR were also negative by the TaqMan RT-PCR. No false positive result was observed for other enteric RNA viruses such as rotavirus and sapovirus. This is the first report on the development of a duplex TaqMan RT-PCR end-point assay for detection and differentiation of GI and GII of NV strains simultaneously followed by genotyping. These results suggest the practical application of this duplex TaqMan RT-PCR is useful for the detection of NV in clinical specimens.
用于检测诺如病毒（NV）的传统逆转录聚合酶链反应（RT-PCR）是一个多步骤的复杂过程，需要凝胶电泳以及杂交或测序来确认最终诊断。开发了一种双重TaqMan RT-PCR来检测和分类NV的基因组（G）I和GII。该检测方法的引物和TaqMan探针选自开放阅读框（ORF）1-ORF2连接区域。本研究使用了来自103起肠胃炎暴发的796份粪便标本，以及一系列包含大多数NV基因型的46份粪便标本。对于来自103起暴发的这些标本，在先前使用传统RT-PCR检测为阳性的541份标本中，有536份通过双重TaqMan RT-PCR检测到NV并进行了分类。在传统RT-PCR检测为阴性的255份标本中，有251份通过TaqMan RT-PCR检测也为阴性。对于其他肠道RNA病毒，如轮状病毒和札如病毒，未观察到假阳性结果。这是关于开发一种双重TaqMan RT-PCR终点检测方法的首次报告，该方法可同时检测和区分NV毒株的GI和GII，并进行基因分型。这些结果表明，这种双重TaqMan RT-PCR在临床标本中检测NV具有实际应用价值。
