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United States Department of Agriculture

Agricultural Research Service

Research Project: MOLECULAR ANALYSIS OF VIRULENCE DETERMINANTS OF SELECT BACTERIA IN FISH DISEASES Title: Simultaneous Detection of Bacterial Fish Pathogens, Edwardsiella ictaluri, Flavobacterium columnnare and Aeromonas Hydrophila by Multiplex-PCR

item Panangala, Victor
item Shoemaker, Craig
item Vansanten, V. - AUBURN UNIVERSITY
item Klesius, Phillip

Submitted to: American Society for Microbiology Annual Meeting
Publication Type: Abstract Only
Publication Acceptance Date: April 30, 2007
Publication Date: May 21, 2007
Citation: Panangala, V.S., Shoemaker, C.A., Vansanten, V.L., Dybvig, K., Klesius, P.H. 2007. Simultaneous Detection of Bacterial Fish Pathogens, Edwardsiella ictaluri, Flavobacterium columnnare and Aeromonas Hydrophila by Multiplex-PCR. American Society for Microbiology Annual Meeting Proceedings. page Z-001.

Technical Abstract: Edwardsiella ictaluri, Flavobacterium columnare and Aeromonas hydrophila are three major bacterial pathogens of fish that cause diseases with significant economic impact on the aquaculture industry world-wide. Rapid detection of multiple infections with these bacteria in the same host is important for prompt implementation of disease control measures. Specific primers for simultaneous amplification of target DNA fragments from F. columnare (504 bp), E. ictaluri (407 bp) and A. hydrophila (209 bp) were used in a multiplex-PCR (m-PCR) assay. The m-PCR was optimized by adjusting the reaction buffers and using a touchdown protocol. Specificity of the m-PCR was assessed with 10 representative isolates of each of the three bacteria and 11 other Gram-negative and 2 Gram-positive bacteria taxonomically related or ubiquitous in the aquatic environment. Blood, gills and kidney tissues from channel catfish (Ictalurus punctatus) experimentally infected with F. columnare, E. ictaluri, and A. hydrophila in different conbinations and tissues spiked with the three bacteria were tested both by m-PCR and bacteriological culture. The lowest detection limit was 20 pg of nucleic acid template from each of the three bacteria per m-PCR reaction mixture. Except for a single species (A. Salmonicida subsp. Salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. The sensitivity threshold for detection of the three bacteria in tissues ranged between 3.4 X 10 to the second power and 2.5 X 10 to the fifth power cells per gram of tissue. The m-PCR is rapid, sensitive and specific for detection of F. columnare, E. ictaluri and A. hydrophila in mixed infections compared to the traditional time-consuming and less reliable bacteriological culture techniques.

Last Modified: 10/31/2014
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