Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida

A real-time PCR assay using a molecular beacon was developed and validated to detect the vapA (surface array protein) gene in the fish pathogen, Aeromonas salmonicida. The assay had 100% analytical specificity and analytical sensitivities of 5 +/- 0fg (DNA), 2.2x104 +/- 1x104CFUg1 (without enrichment) and 40 +/- 10CFUg1 (with enrichment) in kidney tissue. The assay was highly repeatable and proved to be robust following equivalency testing using a different real-time PCR platform. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon, Oncorhynchus tshawytscha (Walbaum), (n=750) and pink shubunkin, Carassius auratus (L.) (n=157). The real-time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for A.salmonicida, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive and a reproducible method for the detection of A.salmonicida. It can be used for diagnostic testing, health certification and active surveillance programmes.