Simultaneous evaluation of four PCR primer sets for the diagnosis of Streptococcus phocae infection

The sensitivity of 4 published primer pairs for the detection of Streptococcus phocae strains was evaluated. Primer sets cpn60-F and -R and sodA-F and -R correctly identified S. phocae. Correct identification was also achieved with the primer pairs cae1-cae2 and PX1-PX2, but using the reverse complementary version of both reverse primers (caeVQ2 and PXVQ2, respectively). Among the 4 PCR protocols with pure and mixed cultures, the primer pair PX1-PXVQ2 provided the highest level of sensitivity for S. phocae (10 ² and 10⁴ cells per PCR tube), and detection was 10- to 100-fold higher than the other 3 primer pairs. When the cae1-caeVQ2 and PX1-PXVQ2 PCR protocols were applied to different seeded Atlantic salmon tissues (spleen, kidney and liver), the detection limit achieved was 5.1 × 10⁵ to 6.4 × 10⁷ CFU g^-1, and the lowest sensitivity detected was 1.18 × 10⁶ S. phocae per tube (which corresponds to 6.4 × 10⁷ CFU g^-1) in spleen samples using PX1-PXVQ2. In the kidney samples seeded with S. phocae strains, regardless of the primer set used, the PCR sensitivity was the same (7.31 ± 1.5 × 10⁶ CFU g^-1). In addition, the nested PCR assay using the primer pair PX1-PXVQ2 improved the sensitivity of detection of S. phocae by at least 100 times compared to the first round PCR, not only in mixed and pure suspensions, but also in experimentally seeded fish tissues. The picked tissues that allowed the easiest detection of S. phocae were the liver, kidney and spleen, respectively. Thus, the nested PCR approach is an important tool for the rapid and reliable diagnosis of streptococcosis due to S. phocae.