title: Application of viability PCR to discriminate the infectivity of hepatitis A virus in food samples.
abstract: Transmitted through the fecal-oral route, the hepatitis A virus (HAV) is acquired primarily through close personal contact and foodborne transmission. HAV detection in food is mainly carried out by quantitative RT-PCR (RT-qPCR). The discrimination of infectious and inactivated viruses remains a key obstacle when using RT-qPCR to quantify enteric viruses in food samples. Initially, viability dyes, propidium monoazide (PMA) and ethidium monoazide (EMA), were evaluated for the detection and quantification of infectious HAV in lettuce wash water. Results showed that PMA combined with 0.5% Triton X-100 (Triton) was the best pretreatment to assess HAV infectivity and completely eliminated the signal of thermally inactivated HAV in lettuce wash water. This procedure was further evaluated in artificially inoculated foods (at concentrations of ca. 6×10(4), 6×10(3) and 6×10(2)TCID50) including lettuce, parsley, spinach, cockles and coquina clams. The PMA-0.5% Triton pretreatment reduced the signal of thermally inactivated HAV between 0.5 and 2 logs, in lettuce and spinach concentrates. Moreover, this pretreatment reduced the signal of inactivated HAV by more than 1.5 logs, in parsley and ten-fold diluted shellfish samples inoculated at the lowest concentration. Overall, this pretreatment (50 μM PMA-0.5% Triton) significantly reduced the detection of thermally inactivated HAV, depending on the initial virus concentration and the food matrix.
authors.affiliation: Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain.
authors.forename: L
authors.initials: L
authors.lastname: Moreno
authors.affiliation: Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain; Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain.
authors.forename: R
authors.initials: R
authors.lastname: Aznar
authors.affiliation: Department of Microbiology and Ecology, University of Valencia, Av. Dr. Moliner, 50, 46100 Burjassot, Valencia, Spain; Department of Biotechnology, Institute of Agrochemistry and Food Technology (IATA-CSIC), Av. Agustín Escardino, 7, 46980 Paterna, Valencia, Spain. Electronic address: gloriasanchez@iata.csic.es.
authors.forename: G
authors.initials: G
authors.lastname: Sánchez
mesh_terms: D000818:Animals; D001386:Azides / Q000737:chemistry; D049872:Bivalvia / Q000821:virology*; D002460:Cell Line; D005516:Food Microbiology / Q000379:methods*; D006506:Hepatitis A / Q000821:virology*; D030041:Hepatitis A virus / Q000235:genetics / Q000502:physiology*; D006358:Hot Temperature; D007202:Indicators and Reagents / Q000032:analysis / Q000592:standards; D050296:Microbial Viability; D017830:Octoxynol / Q000737:chemistry; D011419:Propidium / Q000031:analogs & derivatives / Q000737:chemistry; D012367:RNA, Viral / Q000032:analysis / Q000737:chemistry; D060888:Real-Time Polymerase Chain Reaction / Q000592:standards*; D014675:Vegetables / Q000821:virology*; D038301:Virus Inactivation
chemical_list: D001386:Azides; D007202:Indicators and Reagents; D012367:RNA, Viral; C533957:propidium monoazide; D011419:Propidium; D017830:Octoxynol
keywords: Ethidium monoazide; Hepatitis A virus; Propidium monoazide; Quantitative PCR; Viability dyes