Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida |
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| Authors: | S E Keeling C L Brosnahan C Johnston R Wallis N Gudkovs W L McDonald |
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| Institution: | 1. Animal Health Laboratory, Investigation and Diagnostic Centre – Wallaceville, Ministry for Primary Industries, , Upper Hutt, New Zealand;2. Australian Animal Health Laboratory, AAHL Fish Diseases Laboratory, CSIRO Livestock Industries, , Geelong, Victoria, Australia |
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| Abstract: | 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 ± 0 fg (DNA), 2.2 × 104 ± 1 × 104 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 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. |
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| Keywords: |
Aeromonas salmonicida
molecular beacon
qPCR
real‐time PCR |
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