DAO 97:135-142 (2011)  -  DOI: https://doi.org/10.3354/dao02395

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

D. Tapia-Cammas1, A. Yañez2, G. Arancibia1, A. E. Toranzo3, R. Avendaño-Herrera1,*

1Laboratorio de Patología de Organismos Acuáticos y Biotecnología Acuícola, Facultad de Ciencias Biológicas, Universidad Andrés Bello, Viña del Mar, Chile
2Laboratorio de Enzimología, Instituto de Bioquímica, Facultad de Ciencias, Universidad Austral de Chile, Campus Isla Teja, Casilla 567, Valdivia, Chile
3Departamento de Microbiología y Parasitología, CIBUS, Facultad de Biología e Instituto de Acuicultura, Universidad de Santiago de Compostela 15782, Spain

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 × 105 CFU ml−1 of V. anguillarum, 1.26 × 104 CFU ml−1 of S. phocae, and 5.33 × 104 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 × 107 CFU g−1 for V. anguillarum, 9.03 ± 1.84 × 105 CFU g−1 for S. phocae, 3.8 ± 0.78 × 103 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 simul­taneously 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. 


KEY WORDS: Multiplex PCR · Fish pathogens · Atlantic salmon · Analytic sensitivity test · ­Specificity test


Full text in pdf format 
Cite this article as: Tapia-Cammas D, Yañez A, Arancibia G, Toranzo AE, Avendaño-Herrera R (2011) Multiplex PCR for the detection of Piscirickettsia salmonis, Vibrio anguillarum, Aeromonas salmonicida and Streptococcus phocae in Chilean marine farms. Dis Aquat Org 97:135-142. https://doi.org/10.3354/dao02395

Export citation
Mail this link - Contents Mailing Lists - RSS
- -