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Title: Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water

Author
item REICHLEY, STEPHEN - Mississippi State University
item WARE, CYNTHIA - Mississippi State University
item GREENWAY, TERRENCE - Mississippi State University
item WISE, DAVID - Mississippi State University
item GRIFFIN, MATT - Mississippi State University

Submitted to: Journal of Veterinary Diagnostic Investigation
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 12/8/2014
Publication Date: 3/16/2015
Citation: Reichley, S.R., Ware, C., Greenway, T.E., Wise, D.J., Griffin, M.J. 2015. Real-time PCR assays for detection and quactification of Edwardsiella tarda, Edwardsiella piscicida, Edwardsiella piscicida-like sp. in catfish tissues and pond water. Journal of Veterinary Diagnostic Investigation. 27(2):130-139.

Interpretive Summary: Polymerase chain reaction assays for the detection and quantification Edwardsiella tarda, E. piscicida and E. piscicida-like in catfish tissue, pond water and culture.

Technical Abstract: Researchers have proposed the adoption of 3 distinct genetic taxa among bacteria previously classified as Edwardsiella tarda; namely E. tarda, E. piscicida, and a taxon presently termed E. piscicida–like. Individual real-time polymerase chain reaction (qPCR) assays were developed, based on published primers, for E. tarda, E. piscicida, and E. piscicida–like sp. to provide rapid quantitative confirmatory tests for these phenotypically ambiguous bacteria. The qPCR assays were shown to be repeatable and reproducible, with high degrees of sensitivity and specificity. Each assay showed a linear dynamic range covering 8 orders of magnitude and a sensitivity limit of 5 copies of target DNA in a 15-µL reaction. In addition, each assay was found specific to their respective targets with no observed amplification from nontarget organisms, including the closely related E. ictaluri and E. hoshinae. Under the conditions used in this study, the 3 assays had a quantifiable limit ranging from 103 (E. piscicida) to 102 (E. piscicida–like and E. tarda) colony forming units in kidney tissue biopsies (approximately 25 mg), pond water samples (35 mL), and broth culture (20 µL). In experimental challenges, the assays were able to detect their respective targets in both clinically and subclinically infected channel catfish (Ictalurus punctatus) fingerlings. In addition to quantifying target bacteria from various substrates, the assays provide rapid identification, differentiation, and confirmation of the phenotypically indistinguishable E. tarda, E. piscicida, and E. piscicida–like sp., a valuable tool for diagnostic assessments.