Optimisation of a PMAxx ™ -RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels

AbstractHuman noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate noroviruses in their digestive tissues, are a typical vector for human infection. RT-qPCR, the established method for human norovirus detection in food, does not allow discrimination between infectious and non-infectious viruses and can overestimate potentially infectious viral loads. To develop a more accurate method of infectious norovirus load estimation, we combined intercalating agent propidium monoazide (PMAxx ™)-pre-treatment with RT-qPCR assay using in vitro-cultivable murine norovirus. Three primer sets targeting different genome regions and diverse amplicon sizes were used to compare one-step amplification of a short genome fragment to three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp and 2.3 kbp a mplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated murine noroviruses in PBS suspension, PMAxx™ RT-qPCRs were implemented to detect murine noroviruses subsequent to their extraction from mussel digestive tissues; virus extraction via anionic po lymer-coated magnetic beads was compared with the proteinase K-dependent ISO norm. The long-range RT-qPCR process detecting fragments of more than 2.3 kbp allowed accurate estimation of the infectivity of UV-damaged murine noroviruses. While proteinase K extraction limited later estimation of PMAxx™ pre-treatment effects and was found to be unsuited...
Source: Food and Environmental Virology - Category: Virology Source Type: research