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

Type Article
Date 2021-03
Language English
Author(s) Razafimahefa Ravo M.1, Ludwig-Begall Louisa F.1, Le Guyader Soizick2, Farnir Frédéric3, Mauroy Axel4, Thiry EtienneORCID1
Affiliation(s) 1 : Veterinary Virology and Animal Viral Diseases, Department of Infectious and Parasitic Diseases, FARAH Research Centre, Faculty of Veterinary Medicine, Liège University, B43b, Quartier Vallée 2, Avenue de Cureghem, 10, 4000, Liège, Belgium
2 : Ifremer, Laboratoire de Microbiologie, LSEM-SG2M, BP 21105, 44311, Nantes, France
3 : Biostatistics and Bioinformatics Applied To Veterinary Science, FARAH Research Centre, Faculty of Veterinary Medicine, University of Liège, 4000, Liège, Belgium
4 : Staff Direction for Risk Assessment, Control Policy, Federal Agency for the Safety of the Food Chain, Bld du Jardin Botanique 55, 1000, Brussels, Belgium
Source Food And Environmental Virology (1867-0334) (Springer Science and Business Media LLC), 2021-03 , Vol. 13 , N. 1 , P. 93-106
DOI 10.1007/s12560-020-09454-w
WOS© Times Cited 7
Keyword(s) Norovirus, PMAxx&#8482, Bivalve molluscs, Diagnosis

Human 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 amplicons). 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 polymer-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 to the assay, magnetic bead-captured murine noroviruses retained their infectivity. Genome copies of heat-inactivated murine noroviruses differed by 2.3 log10 between RT-qPCR and PMAxx™-RT-qPCR analysis in bivalve molluscs, the PMAxx™ pre-treatment allowing a closer approximation of infectious titres. The combination of bead-based virus extraction and PMAxx™ RT-qPCR thus provides a more accurate model for the estimation of noroviral bivalve mollusc contamination than the conjunction of proteinase K extraction and RT-qPCR and has the potential (once validated utilising infectious human norovirus) to provide an added measure of security to food safety authorities in the hazard assessment of potential bivalve mollusc contamination.

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Razafimahefa Ravo M., Ludwig-Begall Louisa F., Le Guyader Soizick, Farnir Frédéric, Mauroy Axel, Thiry Etienne (2021). Optimisation of a PMAxx™-RT-qPCR Assay and the Preceding Extraction Method to Selectively Detect Infectious Murine Norovirus Particles in Mussels. Food And Environmental Virology, 13(1), 93-106. Publisher's official version : https://doi.org/10.1007/s12560-020-09454-w , Open Access version : https://archimer.ifremer.fr/doc/00666/77804/