title: Propidium Monoazide Integrated with qPCR Enables the Detection and Enumeration of Infectious Enteric RNA and DNA Viruses in Clam and Fermented Sausages.
abstract: The increase of foodborne viral outbreaks highlights the need for a rapid and sensitive method for the prediction of viral infectivity in food samples. This study assesses the use of propidium monoazide (PMA) coupled with real-time PCR methods (RT-qPCR or qPCR for RNA or DNA viruses, respectively) in the determination of viral infectivity in complex animal-related food matrices. Clam and Spanish fermented sausage ("chorizo") samples were spiked with infectious and heat-inactivated human adenovirus-2 (HAdV-2) and mengovirus (vMC0). PMA-qPCR/RT-qPCR discriminated infective virus particles, with significant reductions (>2.7 log10or 99.7%). Additionally, infectious HAdV-2 and vMC0were quantified by plaque assay (in plaque forming units, PFU), and compared with those in virus genomes copies (GCs) quantified by PMA-qPCR/RT-qPCR. A consistent correlation (R2> 0.92) was showed between PFU and GCs along serial 10-fold dilutions in both DNA and RNA virus and in both food matrices. This study shows the use of PMA coupled to qPCR/RT-qPCR as a promising alternative for prediction of viral infectivity in food samples in comparison to more expensive and time-consuming methods and for those viruses that are not able to grow under available cell culture techniques.
authors.affiliation: Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de Castilla y León Valladolid, Spain.
authors.forename: Narciso M
authors.initials: NM
authors.lastname: Quijada
authors.affiliation: Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina Florianópolis, Brazil.
authors.forename: Gislaine
authors.initials: G
authors.lastname: Fongaro
authors.affiliation: Laboratório de Virologia Aplicada, Departamento de Microbiologia, Imunologia e Parasitologia, Universidade Federal de Santa Catarina Florianópolis, Brazil.
authors.forename: Célia R M
authors.initials: CR
authors.lastname: Barardi
authors.affiliation: Laboratory of Molecular Biology and Microbiology, Instituto Tecnológico Agrario de Castilla y LeónValladolid, Spain; Departamento de Ingeniería Agrícola y Forestal, Tecnología de los Alimentos, Escuela Técnica Superior de Ingenierías Agrarias, Universidad de ValladolidPalencia, Spain.
authors.forename: Marta
authors.initials: M
authors.lastname: Hernández
authors.affiliation: Division of Microbiology, Department of Biotechnology and Food Science, Universidad de Burgos Burgos, Spain.
authors.forename: David
authors.initials: D
authors.lastname: Rodríguez-Lázaro
keywords: PMA-PCR; clam; enteric viruses; fermented sausage; infectivity prediction