1. Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.
- Author
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Keeling SE, Brosnahan CL, Johnston C, Wallis R, Gudkovs N, and McDonald WL
- Subjects
- Aeromonas salmonicida metabolism, Animals, Goldfish, Gram-Negative Bacterial Infections diagnosis, Reproducibility of Results, Salmon, Sensitivity and Specificity, Aeromonas salmonicida classification, Aeromonas salmonicida isolation & purification, Bacterial Proteins metabolism, Fish Diseases diagnosis, Gram-Negative Bacterial Infections veterinary, Real-Time Polymerase Chain Reaction methods
- 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 × 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 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., (© 2012 Blackwell Publishing Ltd.)
- Published
- 2013
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