Monday, April 29, 2013
Norovirus is known to be one of the most prevalent agents for waterborne gastroenteritis world-wide. In developing countries, it is known to cause up to 200,000 deaths annually of children less than 5 years of age. Development of molecular diagnostic methods based on quantitative reverse transcription–Polymerase Chain Reaction (RT-qPCR)–has allowed broader examination of the role of norovirus in epidemic and sporadic gastroenteritis. However, these methods require the essential enzymatic step of reverse transcription which increases the experimental cost, time and complexity. Also, the detection of norovirus is generally limited by factors such as low virus concentrations in sample and inefficient viral RNA extraction methods, particularly for surface waters. Here we report an assay for the detection of norovirus with high sensitivity and specificity using river water. We have utilized the specificity of the Duplex-specific nuclease (DSN), an enzyme originally isolated from the Kamchatka crab, that exhibits a strong preference for cleaving double-stranded DNA or single stranded DNA in a DNA-RNA hybrid duplex. Using the viral genomic RNA as a template, DSN preferrentially cleaves flourescent DNA probes in the DNA-RNA duplex, resulting in an amplified fluorescence signal over time. To demonstrate the efficiency of our assay, we spiked locally collected surface water with norovirus RNA extracted from fecal sources and compared these results against the state of the art detection with RT-qPCR. Our preliminary results show a detection limit of 5-10 norovirus copies per assay, while the RT-qPCR system produces a signal just above threshold, indicating no more than 1 copy for the same sample. Because our method does not require a thermocycler, appears to be more sensitive, and takes less than 30 minutes to complete, it holds great promise for developing countries where sporadic outburst of norovirus and other RNA virus infections can have catastrophic effects.
