Comparison between RT droplet digital PCR and RT real-time PCR for quantification of noroviruses in oysters

Oysters are frequently associated with norovirus outbreaks, but the presence of norovirus RNA in oysters does not necessarily imply a health risk to humans. There is a close link between human illness and consumption of oysters with high levels of norovirus RNA, but oysters with low levels of norovirus RNA are more unlikely to be associated with illness. Reliable and precise quantification methods are therefore important for outbreak investigations and risk assessments. This study optimised and validated RT droplet digital PCR (RT-ddPCR) assays for quantification of norovirus genogroups I and II in artificially contaminated oysters, and compared them with the standard method, RT real-time PCR (RT-qPCR). The two methods had comparable 95% limits of detection, but RT-ddPCR generally showed greater precision in quantification. Differences between fluorometric measurements and quantification with RT-ddPCR were determined on in vitro transcribed RNA with targets for norovirus genogroups I and II. Quantification by RT-ddPCR was on average 100 times lower than the fluorometric value for norovirus GI and 15.8 times lower than the fluorometric value for norovirus GII. The large inter-assay difference observed highlights the need for monitoring the RT efficiency in RT-ddPCR, especially when results from different assays are compared. Overall, this study suggests that RT-ddPCR can be a suitable method for precise quantification of norovirus genogroups I and II in oysters.