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Journal of Clinical Microbiology, August 2006, p. 2714-2720, Vol. 44, No. 8
0095-1137/06/$08.00+0     doi:10.1128/JCM.00443-06
Copyright © 2006, American Society for Microbiology. All Rights Reserved.

Enhanced Reverse Transcription-PCR Assay for Detection of Norovirus Genogroup I

Institut für Laboratoriums und Transfusionsmedizin, Herz und Diabeteszentrum Nordrhein-Westfalen, Universitätsklinik der Ruhr-Universität Bochum, Bad Oeynhausen, Germany,¹ Landesamt für Verbraucherschutz Sachsen-Anhalt, Halle, Germany,² Berliner Betrieb für Zentrale Gesundheitliche Aufgaben (BBGes), Institut für Lebensmittel, Arzneimittel und Tierseuchen (ILAT), Berlin, Germany³

Received 1 March 2006/ Returned for modification 14 May 2006/ Accepted 8 June 2006

We have developed a one-tube reverse transcription (RT)-PCR method using the real-time TaqMan PCR system for the detection of norovirus genogroup I (NV GGI). By introduction of a novel probe based on locked nucleic acid technology, we enhanced the sensitivity of the assay compared to those of conventional TaqMan probes. The sensitivity of the NV GGI RT-PCR was determined by probit analysis with defined RNA standards and quantified norovirus isolates to 711 copies/ml (95% detection limit). In order to detect PCR inhibition, we included a heterologous internal control (IC) system based on phage MS2. This internally controlled RT-PCR was tested on different real-time PCR platforms, LightCycler, Rotorgene, Mastercycler EP realplex, and ABI Prism. Compared to the assay without an IC, the duplex RT-PCR exhibited no reduction in sensitivity in clinical samples. In combination with an established NV GGII real-time RT-PCR, we used the novel assay in a routine assay for diagnosis of clinical and food-borne norovirus infection. We applied this novel assay to analyze outbreaks of nonbacterial acute gastroenteritis. Norovirus of GGI was detected in these outbreaks. Sequence and similarity plot analysis of open reading frame 1 (ORF1) and ORF2 showed two genotypes, GGI/2 and GGI/4, in semiclosed communities.