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Sensitive detection of water- and foodborne enteric viruses is extremely relevant, especially due to the low concentrations in which they are found. Accurate and sensitive detection of Norovirus, the primary responsible for water- and foodborne outbreaks, is of particular importance. Quantification of Norovirus is commonly performed by quantitative RT-PCR (RT-qPCR). In recent years a new platform was developed, digital PCR, that quantifies without the need for a standard curve thus decreasing the errors associated with its utilization. The platform developed by LifeTechnologies, QuantStudio 3D Digital PCR is amongst the least studied digital platform and although it allows the direct detection of DNA targets it requires a two-step RT-PCR for the detection of RNA targets. In this work we developed a new protocol able to detect Norovirus using a one-step digital PCR reaction (RT-dPCR). The performance of the newly developed one-step digital PCR was compared to RT-qPCR for the detection of Norovirus genogroup I and genogroup II. The sensitivity of RT-dPCR was identical to that of RT-qPCR, and the quantitative data determined by both methods were not significantly different for most samples. This one-step absolute quantification approach is a useful tool to minimize the time spent currently using this particular platform to amplify viral RNA and to standardize quantification of enteric viruses in food and environmental samples. This study proved the usefulness of the newly developed RT-dPCR protocol for a sensitive and accurate detection of low-copy targets.

Nanofluidic digital PCR for the quantification of Norovirus for water quality assessment