Novel LUX^TM Real-time PCR Assays for Detection and Quantification of Genogroups I and II Noroviruses in Clinical Specimens

Division of Molecular Virology, Department of Molecular and Clinical Medicine, Linköping University, Sweden; Department of Microbiology, University of León (UNAN), Nicaragua; Microbiological Laboratory, Ryhov County Hospital, Jönköping, Sweden; Division of Medical Microbiology, Department of Molecular and Clinical Medicine, Linköping University, Sweden

^* To whom correspondence should be addressed. Email: perli{at}imk.liu.se.

	Abstract

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^TM (LUX^TM) real-time PCR assays for detection and quantification of norovirus genogroups I and II. The LUX^TM 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^TM 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.