title: Nested multiplex PCR assay for detection of human enteric viruses in shellfish and sewage.
abstract: Environmental samples and contaminated shellfish present frequently low concentrations of more than one viral species. For this reason, a nested multiplex RT-PCR was developed for the detection of adenoviruses, enteroviruses and hepatitis A viruses in different environmental samples such as urban sewage and shellfish. This assay will save time and cost for detection of these enteric viruses with a smaller sample volume, which otherwise can be a limiting factor in routine analysis. The limit of detection was approximately 1 copy for adenovirus and 10 copies for enterovirus and hepatitis A virus per PCR reaction using titrated cell-cultured viruses as template material. In shellfish and environmental samples, this multiplex PCR was optimized to detect all three viruses simultaneously when the concentration of each virus was equal or lower than 1000 copies per PCR reaction. This is the level found predominantly in the environment and in shellfish when the numbers of fecal bacterial and phage indicators are low. The detection of human adenoviruses by PCR has been suggested as a molecular index of fecal contamination of human origin in the environment and food and the multiplex assay developed may be a tool for evaluating the presence of viral contamination in shellfish and water and to expand microbiological control to include viral markers.
authors.affiliation: Department of Microbiology, Faculty of Biology, University of Barcelona, Av. Diagonal 645, 08028 Barcelona, Spain.
authors.forename: Meritxell
authors.initials: M
authors.lastname: Formiga-Cruz
authors.forename: Ayalkibet
authors.initials: A
authors.lastname: Hundesa
authors.forename: Pilar
authors.initials: P
authors.lastname: Clemente-Casares
authors.forename: Nestor
authors.initials: N
authors.lastname: Albiñana-Gimenez
authors.forename: Annika
authors.initials: A
authors.lastname: Allard
authors.forename: Rosina
authors.initials: R
authors.lastname: Girones
mesh_terms: D000260:Adenoviruses, Human / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D000818:Animals; D004770:Enterovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D006507:Hepatovirus / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification; D006801:Humans; D016133:Polymerase Chain Reaction / Q000379:methods*; D012680:Sensitivity and Specificity; D012722:Sewage / Q000821:virology*; D012758:Shellfish / Q000821:virology*; D014871:Water Microbiology
chemical_list: D012722:Sewage