Fast detection of Noroviruses using a real-time PCR assay and automated sample preparation

We have designed a new real-time RT-PCR assay on the LightCycler (LC) with SYBR Green detection and melting curve analysis (Tm) to detect NoV RNA in patient stool samples. The performance of the real-time PCR assay was compared with that obtained in parallel with a commercially available enzyme immunoassay (ELISA) for antigen detection by testing a panel of 52 stool samples. Additionally, in a collaborative study with the Baden-Wuerttemberg State Health office, Stuttgart (Germany) the real-time PCR results were blindly assessed using a previously well-established nested PCR (nPCR) as the reference method, since PCR-based techniques are now considered as the "gold standard" for NoV detection in stool specimens.Analysis of 52 clinical stool samples by real-time PCR yielded results that were consistent with reference nPCR results, while marked differences between the two PCR-based methods and antigen ELISA were observed. Our results indicate that PCR-based procedures are more sensitive and specific than antigen ELISA for detecting NoV in stool specimens.The combination of automated sample preparation and real-time PCR provided reliable diagnostic results in less time than conventional RT-PCR assays. These benefits make it a valuable tool for routine laboratory practice especially in terms of rapid and appropriate outbreak-control measures in health-care facilities and other settings.NoV, members of the family Caliciviridae, are single-stranded RNA, nonenveloped viruses. Based on antigenic and genetic distinctions NoV (formerly called Norwalk-like viruses) can be divided in 5 different genogroups [1]. Since the first identification of this pathogen in 1972 [2], NoV have become one of the most commonly reported causative agents of large outbreaks of non-bacterial gastroenteritis worldwide as well as sporadic gastroenteritis in the community [3-6]. NoV usually cause acute self-limited infections in humans of all ages. However, NoV infection can be severe in frail elderly