title: Development and validation of real-time PCR for the detection of Yersinia ruckeri.
abstract: Yersiniosis (enteric red mouth disease) is a contagious bacterial disease caused by Yersinia ruckeri, which primarily affects salmonids. A real-time PCR assay using a molecular beacon has been developed and validated to improve the detection of the causative biotypes of Y. ruckeri. The assay, which targets the glnA (glutamine synthetase) gene, proved to have 100% analytical specificity and analytical sensitivities of 5 fg and 3 × 10(3) CFU g(-1) for DNA and seeded kidney tissue, respectively. The assay was highly repeatable with low % CV for intra- and inter-run experiments, and the optimized parameters transferred easily between different real-time PCR platforms. Following analytical validation, diagnostic specificity was determined using New Zealand farmed Chinook salmon (n = 750) from 10 farms during 2007/08. The real-time PCR was run in parallel with the bacterial culture detection method, and all fish tested were found to be negative by both methods for Y. ruckeri, resulting in 100% diagnostic specificity (95% confidence interval). The molecular beacon real-time PCR system is specific, sensitive, reproducible and a rapid method for the detection of Y. ruckeri and has the potential to be used for routine diagnostic testing, health certification and active surveillance programmes.
authors.affiliation: Animal Health Laboratory, Investigation and Diagnostic Centre - Wallaceville, MAF Biosecurity New Zealand, Upper Hutt, New Zealand. suzanne.keeling@maf.govt.nz
authors.forename: S E
authors.initials: SE
authors.lastname: Keeling
authors.forename: C
authors.initials: C
authors.lastname: Johnston
authors.forename: R
authors.initials: R
authors.lastname: Wallis
authors.forename: C L
authors.initials: CL
authors.lastname: Brosnahan
authors.forename: N
authors.initials: N
authors.lastname: Gudkovs
authors.forename: W L
authors.initials: WL
authors.lastname: McDonald
mesh_terms: D000818:Animals; D005393:Fish Diseases / Q000175:diagnosis*; D005974:Glutamate-Ammonia Ligase / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000662:veterinary*; D015203:Reproducibility of Results; D012487:Salmonidae / Q000382:microbiology*; D012680:Sensitivity and Specificity; D015009:Yersinia Infections / Q000175:diagnosis / Q000662:veterinary*; D044124:Yersinia ruckeri / Q000235:genetics* / Q000302:isolation & purification
chemical_list: D005974:Glutamate-Ammonia Ligase