Premium
Development and validation of a real‐time PCR assay for the detection of A eromonas salmonicida
Author(s) -
Keeling S E,
Brosnahan C L,
Johnston C,
Wallis R,
Gudkovs N,
McDonald W L
Publication year - 2013
Publication title -
journal of fish diseases
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.819
H-Index - 85
eISSN - 1365-2761
pISSN - 0140-7775
DOI - 10.1111/jfd.12014
Subject(s) - real time polymerase chain reaction , biology , aeromonas salmonicida , pathogen , microbiology and biotechnology , fish <actinopterygii> , chromatography , gene , fishery , chemistry , genetics
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.