Rapid Detection and Quantification of Norovirus in Environmental Waters Using a Novel Isothermal Assay

Norovirus is one of the most prevalent agents for waterborne gastroenteritis and is known to cause a large number of deaths annually. Molecular diagnostic methods based on Reverse Transcription–Polymerase Chain Reaction (RT-PCR) are costly and limited by the essential enzymatic step of reverse transcription. Here we report an assay for the detection of norovirus with high sensitivity and specificity. We use the specificity of the Duplex-specific nuclease (DSN), an enzyme that exhibits a strong preference for cleaving single-stranded DNA in a DNA-RNA hybrid duplex. Using the viral genomic RNA as a template, DSN preferentially cleaves fluorescent DNA probes in the DNA-RNA duplex, resulting in an amplified fluorescence signal. We spiked environmental with norovirus RNA and compared the assay against RT-PCR. Our preliminary results show a detection limit of 10 norovirus copies per assay, which is better than RT-PCR.