title: Evaluation of viability PCR performance for assessing norovirus infectivity in fresh-cut vegetables and irrigation water.
abstract: Norovirus (NoV) detection in food and water is mainly carried out by quantitative RT-PCR (RT-qPCR). The inability to differentiate between infectious and inactivated viruses and the resulting overestimation of viral targets is considered a major disadvantage of RT-qPCR. Initially, conventional photoactivatable dyes (i.e. propidium monoazide, PMA and ethidium monoazide, EMA) and newly developed ones (i.e. PMAxx and PEMAX) were evaluated for the discrimination between infectious and thermally inactivated NoV genogroup I (GI) and II (GII) suspensions. Results showed that PMAxx was the best photoactivatable dye to assess NoV infectivity. This procedure was further optimized in artificially inoculated lettuce. Pretreatment with 50μM PMAxx and 0.5% Triton X-100 (Triton) for 10min reduced the signal of thermally inactivated NoV by ca. 1.8 logs for both genogroups in lettuce concentrates. Additionally, this pretreatment reduced the signal of thermally inactivated NoV GI between 1.4 and 1.9 logs in spinach and romaine and lamb's lettuces and by >2 logs for NoV GII in romaine and lamb's lettuce samples. Moreover this pretreatment was satisfactorily applied to naturally-contaminated water samples with NoV GI and GII. Based on the obtained results this pretreatment has the potential to be integrated in routine diagnoses to improve the interpretation of positive NoV results obtained by RT-qPCR.
authors.affiliation: Department of Agricultural and Forest Science, University of Palermo, Palermo, Italy; Biotechnology Department, (IATA-CSIC), Av. Agustín Escardino 7, Paterna, 46980 Valencia, Spain.
authors.forename: W
authors.initials: W
authors.lastname: Randazzo
authors.affiliation: Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo, 25, 30100 Murcia, Spain.
authors.forename: Francisco
authors.initials: F
authors.lastname: López-Gálvez
authors.affiliation: Research Group on Quality, Safety and Bioactivity of Plant Foods, Department of Food Science and Technology, CEBAS-CSIC, Campus Universitario de Espinardo, 25, 30100 Murcia, Spain.
authors.forename: A
authors.initials: A
authors.lastname: Allende
authors.affiliation: Biotechnology Department, (IATA-CSIC), Av. Agustín Escardino 7, Paterna, 46980 Valencia, Spain; Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, Burjassot, 46100 Valencia, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Aznar
authors.affiliation: Biotechnology Department, (IATA-CSIC), Av. Agustín Escardino 7, Paterna, 46980 Valencia, Spain; Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, Burjassot, 46100 Valencia, Spain. Electronic address: gloriasanchez@iata.csic.es.
authors.forename: G
authors.initials: G
authors.lastname: Sánchez
mesh_terms: D005502:Food / Q000821:virology*; D064088:Hazard Analysis and Critical Control Points / Q000379:methods*; D050296:Microbial Viability; D029322:Norovirus / Q000235:genetics / Q000502:physiology*; D012367:RNA, Viral / Q000032:analysis; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D014675:Vegetables / Q000821:virology*; D038301:Virus Inactivation; D014871:Water Microbiology
chemical_list: D012367:RNA, Viral
keywords: Irrigation water; Norovirus; Photoactivatable dyes; Propidium monoazide; Quantitative RT-PCR