Development and validation of a real‐time PCR assay for the detection of Aeromonas salmonicida. Issue 5 (5th November 2012)

• Record Type: Journal Article
• Authors: Keeling, S E; Brosnahan, C L; Johnston, C; Wallis, R; Gudkovs, N; McDonald, W L
• Is Part Of: Journal of fish diseases. Volume 36:Issue 5(2013:May)
• Page Start: 495
• Page End: 503
• Publication Date: 2012-11-05
• Subjects: Fishes -- Diseases -- Periodicals; 639.3
• DOI: 10.1111/jfd.12014
• Languages: English
• ISSNs: 0140-7775

Abstract

<abstract abstract-type="main" id="jfd12004-abs-0001"> <title>Abstract</title> <p>A real‐time PCR assay using a molecular beacon was developed and validated to detect the <italic>vapA</italic> (surface array protein) gene in the fish pathogen, <italic>Aeromonas salmonicida</italic>. 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, <italic>Oncorhynchus tshawytscha</italic> (Walbaum), (<italic>n</italic> = 750) and pink shubunkin, <italic>Carassius auratus</italic> (L.) (<italic>n</italic> = 157). The real‐time PCR was run in parallel with culture and all fish tested were found to be negative by both methods for <italic>A. salmonicida</italic>, 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 <italic>A. salmonicida</italic>. It can be used for diagnostic testing, health certification and active surveillance programmes.</p> </abstract>