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

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