Development and validation of a real‐time PCR assay for the detection of A eromonas 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, A eromonas salmonicida . The assay had 100% analytical specificity and analytical sensitivities of 5 ± 0 fg ( DNA ), 2.2 × 10 4  ± 1 × 10 4  CFU g −1 (without enrichment) and 40 ± 10 CFU g −1 (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 N ew Z ealand farmed C hinook salmon, O ncorhynchus tshawytscha ( W albaum), ( n  = 750) and pink shubunkin, C arassius 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.