Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

D. Tapia-Cammas, A. Yañez, G. Arancibia, A. E. Toranzo, R. Avendaño-Herrera

Resultado de la investigación: Article

13 Citas (Scopus)

Resumen

A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg μl -1 for V. anguillarum, 500 fg μl -1 for P. salmonis, and 5 pg μl -1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml -1 of V. anguillarum, 1.26 × 10 4 CFU ml -1 of S. phocae, and 5.33 × 10 4 CFU ml -1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g -1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g -1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg -1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.

Idioma originalEnglish
Páginas (desde-hasta)135-142
Número de páginas8
PublicaciónDiseases of Aquatic Organisms
Volumen97
N.º2
DOI
EstadoPublished - 6 dic 2011

Huella dactilar

Streptococcus phocae
Piscirickettsia salmonis
Aeromonas salmonicida
Vibrio anguillarum
farm
farms
fish
pathogen
DNA
salmon
amplification
detection limit
muscle
assay
gene
pathogens
DNA primers
detection
spleen
kidneys

ASJC Scopus subject areas

  • Aquatic Science
  • Ecology, Evolution, Behavior and Systematics

Citar esto

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title = "Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms",
abstract = "A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg μl -1 for V. anguillarum, 500 fg μl -1 for P. salmonis, and 5 pg μl -1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml -1 of V. anguillarum, 1.26 × 10 4 CFU ml -1 of S. phocae, and 5.33 × 10 4 CFU ml -1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g -1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g -1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg -1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.",
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Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms. / Tapia-Cammas, D.; Yañez, A.; Arancibia, G.; Toranzo, A. E.; Avendaño-Herrera, R.

En: Diseases of Aquatic Organisms, Vol. 97, N.º 2, 06.12.2011, p. 135-142.

Resultado de la investigación: Article

TY - JOUR

T1 - Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms

AU - Tapia-Cammas, D.

AU - Yañez, A.

AU - Arancibia, G.

AU - Toranzo, A. E.

AU - Avendaño-Herrera, R.

PY - 2011/12/6

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N2 - A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg μl -1 for V. anguillarum, 500 fg μl -1 for P. salmonis, and 5 pg μl -1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml -1 of V. anguillarum, 1.26 × 10 4 CFU ml -1 of S. phocae, and 5.33 × 10 4 CFU ml -1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g -1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g -1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg -1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.

AB - A multiplex (m-)PCR-based protocol was designed for the simultaneous detection of the main marine bacterial pathogens in Chilean salmon farms: Streptococcus phocae, Aeromonas salmonicida, Vibrio anguillarum and Piscirickettsia salmonis. Each of the 4 oligonucleotide primer pairs exclusively amplified the target gene of the specific bacterial pathogen. The detection limit of the m-PCR using purified total bacterial DNA was 50 pg μl -1 for V. anguillarum, 500 fg μl -1 for P. salmonis, and 5 pg μl -1 for S. phocae and A. salmonicida. This corresponded to average limits in the m-PCR sensitivity of 3.69 × 10 5 CFU ml -1 of V. anguillarum, 1.26 × 10 4 CFU ml -1 of S. phocae, and 5.33 × 10 4 CFU ml -1 of A. salmonicida, while the detection limits for the spiked fish tissues, regardless of the sample (spleen, kidney, liver or muscle) were 2.64 ± 0.54 × 10 7 CFU g -1 for V. anguillarum, 9.03 ± 1.84 × 10 5 CFU g -1 for S. phocae, 3.8 ± 0.78 × 10 3 CFU mg -1 for A. salmonicida and 100 P. salmonis cells. However, high amounts of DNA from 3 bacterial species had a reduction of ~1 log-unit on the amplification sensitivity of S. phocae or A. salmonicida when these were present in lower concentration in the multiplex reaction. The assay described in this study is a rapid, sensitive and efficient tool to detect the presence of S. phocae, A. salmonicida, V. anguillarum and P. salmonis simul taneously from pure cultures and tissues from clinically diseased fish. Therefore, it may be a useful alternative to culture-based methods for the diagnosis of infections in fish obtained from Chilean salmon farms.

KW - Analytic sensitivity test

KW - Atlantic salmon

KW - Fish pathogens

KW - Multiplex PCR

KW - Specificity test

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