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Novel Light-Upon-Extension Real-Time PCR Assays for Detection and Quantification of Genogroup I and II Noroviruses in Clinical Specimens

Division of Molecular Virology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden,¹ Department of Microbiology, University of León, León, Nicaragua,² Microbiological Laboratory, Ryhov County Hospital, Jönköping, Sweden,³ Division of Medical Microbiology, Department of Clinical and Experimental Medicine, Linköping University, Linköping, Sweden⁴

Received 1 July 2007/ Returned for modification 31 August 2007/ Accepted 16 October 2007

Norovirus is now recognized as the leading cause of nonbacterial acute gastroenteritis in adults, causing numerous outbreaks worldwide. We have developed two novel light-upon-extension (LUX) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX system uses a fluorophore attached to one primer having a self-quenching hairpin structure, making it cost-effective and specific. The assays were evaluated against clinical stool specimens (n = 103) from Sweden and Nicaragua and compared to established methods. The norovirus assay detected more positive stool specimens (47/103) than conventional PCR (39/103) and corresponded to a TaqMan real-time PCR, with the exception of one specimen. Furthermore, the assays correctly identified all (n = 11) coded control specimens in a reference panel containing various genogroups and genotypes. Both LUX real-time PCR assays had a wide dynamic range, detecting from 10¹ to 10⁷ genes per reaction, resulting in a theoretical lower limit of 20 000 viruses per gram of stool. No cross-reactivity was noticed with specimens containing other enteric viruses, and by using melting curve analysis we could differentiate between norovirus genogroups I and II.

Published ahead of print on 24 October 2007.