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

Abstract

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 (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 ≤10 1 to 10 7 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.