title: Fast detection of Vibrio species potentially pathogenic for mollusc.
abstract: Vibrio tasmaniensis, Vibrio splendidus and Vibrio neptunius species were distributed worldwide and associated with aquaculture and have been reported as the cause of diseases in aquatic organisms. Polyphasic analyses for bacterial identification are not feasible for routine diagnostic because of the time involved. The aim of this study is to design three PCR primer sets that can assist with fast detection of these species. They were designed from the 16S ribosomal RNA gene, and PCR conditions were found. Each PCR test successfully identified all the tested strains of each target species. The combined specificity of V. tasmaniensis and V. splendidus primer sets offered the best coverage (86%) in terms of separating target organisms from other related species. The primer set of V. tasmaniensis showed a lower sensitivity limit (500 fg of DNA) than the V. splendidus set (1 pg) and both sets gave positive amplification using homogenized tissues from inoculated clams, with 10(2) and 10(4) cfu/g of clam, respectively. The primer set of V. neptunius was highly specific, showing only cross-reaction with V. parahaemolyticus species from 44 tested species. Its sensitivity limit was 100 pg of DNA. A small number of biochemical tests were proposed concurrently with the PCR to differentiate the cross-reacting bacteria. The time of detection of the three tested species was reduced and the further affected animals can be diagnosed in a rapid fraction of time. The detection of virulent strains of V. tasmaniensis pointed to the risk of mollusc culture outbreaks.
authors.affiliation: Area de Microbiología, Departamento de Biología Funcional y Ciencias de la Salud, Facultad de Biología, Universidad de Vigo, Lagoas Marcosende s/n, 36310 Vigo, Spain.
authors.forename: Estela Pérez
authors.initials: EP
authors.lastname: Lago
authors.forename: Teresa Pérez
authors.initials: TP
authors.lastname: Nieto
authors.forename: Rosa
authors.initials: R
authors.lastname: Farto Seguín
mesh_terms: D000818:Animals; D017756:Aquaculture; D015373:Bacterial Typing Techniques; D049872:Bivalvia / Q000382:microbiology*; D017931:DNA Primers / Q000235:genetics; D004269:DNA, Bacterial / Q000235:genetics; D004275:DNA, Ribosomal / Q000235:genetics; D008969:Molecular Sequence Data; D010125:Ostreidae / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014733:Vibrio / Q000145:classification / Q000235:genetics / Q000302:isolation & purification*
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D004275:DNA, Ribosomal; D012336:RNA, Ribosomal, 16S