Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels
نویسندگان
چکیده
Human noroviruses are a major cause for gastroenteritis outbreaks. Filter-feeding bivalve molluscs, which accumulate in their digestive tissues, typical vector human infection. RT-qPCR, the established method norovirus detection food, does not allow discrimination between infectious and non-infectious viruses can overestimate potentially viral loads. To develop more accurate of load estimation, we combined intercalating agent propidium monoazide (PMAxx™)-pre-treatment with RT-qPCR assay using vitro-cultivable murine norovirus. Three primer sets targeting different genome regions diverse amplicon sizes were used to compare one-step amplification short fragment three two-step long-range RT-qPCRs (7 kbp, 3.6 kbp 2.3 amplicons). Following initial assays performed on untreated infectious, heat-, or ultraviolet-inactivated PBS suspension, PMAxx™ implemented detect subsequent extraction from mussel tissues; virus via anionic polymer-coated magnetic beads was compared proteinase K-dependent ISO norm. The process detecting fragments than allowed estimation infectivity UV-damaged noroviruses. While K limited later pre-treatment effects found be unsuited assay, bead-captured retained infectivity. Genome copies heat-inactivated differed by log10 PMAxx™-RT-qPCR analysis allowing closer approximation titres. combination bead-based thus provides model noroviral mollusc contamination conjunction has potential (once validated utilising norovirus) provide an added measure security food safety authorities hazard assessment contamination.
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ژورنال
عنوان ژورنال: Food and Environmental Virology
سال: 2021
ISSN: ['1867-0342', '1867-0334']
DOI: https://doi.org/10.1007/s12560-020-09454-w