PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

R. Avendaño-Herrera, J. P. Maldonado, D. Tapia-Cammas, C. G. Feijóo, F. Calleja, A. E. Toranzo

Resultado de la investigación: Article

11 Citas (Scopus)

Resumen

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 × 10 3 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.

Idioma originalEnglish
Páginas (desde-hasta)223-234
Número de páginas12
PublicaciónDiseases of Aquatic Organisms
Volumen107
N.º3
DOI
EstadoPublished - 16 ene 2014

Huella dactilar

Vibrio ordalii
hemolysins
polymerase chain reaction
amplification
gene
genes
fish
vibriosis
salmonid
agar
Salmo salar
aquaculture
muscle
protocol
detection
cross reaction
diagnostic techniques
disease control
cages
spleen

ASJC Scopus subject areas

  • Aquatic Science
  • Ecology, Evolution, Behavior and Systematics

Citar esto

Avendaño-Herrera, R. ; Maldonado, J. P. ; Tapia-Cammas, D. ; Feijóo, C. G. ; Calleja, F. ; Toranzo, A. E. / PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene. En: Diseases of Aquatic Organisms. 2014 ; Vol. 107, N.º 3. pp. 223-234.
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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 × 10 3 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.",
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PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene. / Avendaño-Herrera, R.; Maldonado, J. P.; Tapia-Cammas, D.; Feijóo, C. G.; Calleja, F.; Toranzo, A. E.

En: Diseases of Aquatic Organisms, Vol. 107, N.º 3, 16.01.2014, p. 223-234.

Resultado de la investigación: Article

TY - JOUR

T1 - PCR protocol for detection of Vibrio ordalii by amplification of the vohB (hemolysin) gene

AU - Avendaño-Herrera, R.

AU - Maldonado, J. P.

AU - Tapia-Cammas, D.

AU - Feijóo, C. G.

AU - Calleja, F.

AU - Toranzo, A. E.

PY - 2014/1/16

Y1 - 2014/1/16

N2 - 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 × 10 3 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.

AB - 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 × 10 3 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.

KW - Atypical vibriosis

KW - Diagnosis

KW - PCR

KW - Salmonid fish

KW - Vibrio ordalii

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U2 - 10.3354/dao02684

DO - 10.3354/dao02684

M3 - Article

C2 - 24429473

AN - SCOPUS:84893448718

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SP - 223

EP - 234

JO - Diseases of Aquatic Organisms

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