The validation and utility of a quantitative one-step multiplex RT real-time PCR targeting Rotavirus A and Norovirus

► A multiplex real-time PCR was developed for the detection and quantitation of Rotavirus A and Norovirus genogroup II. ► An internal extraction and amplification control was incorporated. ► Real-time PCR was compared to the current gold standard, enzyme immunoassay. ► Real-time PCR was significantly more sensitive than enzyme immunoassay. ► Quantitation demonstrated that the viral loads of both pathogens were ten times greater in stools children with diarrhea than in children without diarrhea.