Environmental Detection of Genogroup I, II, and IV Noroviruses by Using a Generic Real-Time Reverse Transcription-PCR Assay

Norovirus is the most common agent implicated in food-borne outbreaks and is frequently detected in environmental samples. These viruses are highly diverse, and three genogroups (genogroup I [GI], GII, and GIV) infect humans. Being noncultivable viruses, real-time reverse transcription-PCR (RT-PCR) is the only sensitive method available for their detection in food or environmental samples. Selection of consensus sequences for the design of sensitive assays has been challenging due to sequence diversity and has led to the development of specific real-time RT-PCR assays for each genogroup. Thus, sample screening can require several replicates for amplification of each genogroup (without considering positive and negative controls or standard curves). This study reports the development of a generic assay that detects all three human norovirus genogroups on a qualitative basis using a one-step real-time RT-PCR assay. The generic assay achieved good specificity and sensitivity for all three genogroups, detected separately or in combination. At variance with multiplex assays, the choice of the same fluorescent dye for all three probes specific to each genogroup allows the levels of fluorescence to be added and may increase assay sensitivity when multiple strains from different genogroups are present. When it was applied to sewage sample extracts, this generic assay successfully detected norovirus in all samples found to be positive by the genogroup-specific RT-PCRs. The generic assay also identified all norovirus-positive samples among 157 archived nucleic acid shellfish extracts, including samples contaminated by all three genogroups.