title: Development and validation of a real-time PCR assay for the detection of Aeromonas salmonicida.
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.
authors.affiliation: Animal Health Laboratory, Investigation and Diagnostic Centre - Wallaceville, Ministry for Primary Industries, Upper Hutt, New Zealand. suzanne.keeling@mpi.govt.nz
authors.forename: S E
authors.initials: SE
authors.lastname: Keeling
authors.forename: C L
authors.initials: CL
authors.lastname: Brosnahan
authors.forename: C
authors.initials: C
authors.lastname: Johnston
authors.forename: R
authors.initials: R
authors.lastname: Wallis
authors.forename: N
authors.initials: N
authors.lastname: Gudkovs
authors.forename: W L
authors.initials: WL
authors.lastname: McDonald
mesh_terms: D048409:Aeromonas salmonicida / Q000145:classification* / Q000302:isolation & purification* / Q000378:metabolism; D000818:Animals; D001426:Bacterial Proteins / Q000378:metabolism*; D005393:Fish Diseases / Q000175:diagnosis*; D006054:Goldfish; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000662:veterinary*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D015203:Reproducibility of Results; D012474:Salmon; D012680:Sensitivity and Specificity
chemical_list: D001426:Bacterial Proteins