title: PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene.
abstract: Vibrio ordalii is the causative agent of atypical vibriosis and has the potential to cause severe losses in salmonid aquaculture. To prevent and control outbreaks, a rapid, reproducible, sensitive, and effective diagnostic method is needed. We evaluated a new conventional polymerase chain reaction (PCR) and real-time PCR (qPCR) protocol using a primer set (VohB_Fw-VohB_Rv) designed to amplify a 112 bp fragment flanking the vohB gene (coding for hemolysin production), against 24 V. ordalii strains isolated from different fish species, the V. ordalii type strain, and 42 representative related and unrelated bacterial species. The primer set was species-specific, recognizing all V. ordalii strains evaluated, with no cross-reaction with the other bacterial species. A sensitivity of 103 copies of the vohB gene was obtained with a standard curve. When the VohB_Fw-VohB_Rv qPCR protocol was applied to Atlantic salmon seeded tissues (kidney, liver, spleen, and muscle), the detection limit ranged from 5.27 × 102 to 4.13 × 103 V. ordalii CFU ml-1, i.e. 62 to 145 copies of the vohB gene, using the previously calculated standard curve. The conventional PCR also detected V. ordalii, but the total reaction time was 1 h longer. When the qPCR protocol was applied to naturally infected cage-cultured Atlantic salmon samples, 5 of 8 fish tested positive for V. ordalii, but only one of them was diagnosed as positive by direct cultivation on agar. We conclude that the PCR protocol evaluated is fast, specific, and sensitive enough to detect V. ordalii in infected tissues and is an important tool for secure diagnosis of atypical vibriosis, and is therefore helpful for the control of the disease through the prompt detection within fish populations.
authors.affiliation: Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Departamento de Ciencias Biológicas, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile.
authors.forename: R
authors.initials: R
authors.lastname: Avendaño-Herrera
authors.forename: J P
authors.initials: JP
authors.lastname: Maldonado
authors.forename: D
authors.initials: D
authors.lastname: Tapia-Cammas
authors.forename: C G
authors.initials: CG
authors.lastname: Feijóo
authors.forename: F
authors.initials: F
authors.lastname: Calleja
authors.forename: A E
authors.initials: AE
authors.lastname: Toranzo
mesh_terms: D000818:Animals; D015964:Gene Expression Regulation, Bacterial / Q000502:physiology*; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism*; D007668:Kidney / Q000382:microbiology; D008099:Liver / Q000382:microbiology; D018482:Muscle, Skeletal / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000379:methods*; D020317:Salmo salar; D012680:Sensitivity and Specificity; D013154:Spleen / Q000382:microbiology; D046509:Tissue Culture Techniques; D014733:Vibrio / Q000145:classification* / Q000235:genetics*
chemical_list: D006460:Hemolysin Proteins