A SYBR Green RT-PCR assay in single tube to detect human and bovine noroviruses and control for inhibition

BACKGROUND: Noroviruses are single-stranded RNA viruses belonging to the family Caliciviridae. They are a major cause of epidemic and sporadic gastroenteritis in humans and clinical signs and lesions of gastroenteritis were reported in bovines. Due to their genetic proximity, potential zoonotic transmission or animal reservoir can be hypothesized for noroviruses. RT-PCR has become the "gold standard" for the detection of noroviruses in faecal and environmental samples. With such samples, the control for inhibition of the reaction during amplification and detection is crucial to avoid false negative results, which might otherwise not be detected. The aim of the reported method is to detect, with a SYBR Green technology, a broad range of noroviruses with a control for inhibition. RESULTS: A SYBR Green real-time RT-PCR assay was developed making use of a foreign internal RNA control added in the same tube. This assay is able to detect human and bovine noroviruses belonging to genogroups I, II and III and to distinguish between norovirus and internal control amplicons using melting curve analysis. A 10-fold dilution of samples appears to be the method of choice to remove inhibition. This assay was validated with human and bovine stool samples previously tested for norovirus by conventional RT-PCR. CONCLUSION: This SYBR Green real-time RT-PCR assay allows the detection of the most important human and bovine noroviruses in the same assay, and avoids false negative results making use of an internal control. Melting curves allow the discrimination between the internal control and norovirus amplicons. It gives preliminary information about the species of origin. The sensitivity of the developed assay is higher than conventional RT-PCR and a 10-fold dilution of samples showed a better efficiency and reproducibility to remove RT-PCR inhibition than addition of bovine serum albumin.