DAO 74:199-208 (2007)  -  doi:10.3354/dao074199

Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila

Victor S. Panangala1,*, Craig A. Shoemaker1, Vicky L. van Santen2, Kevin Dybvig3, Phillip H. Klesius1

1Aquatic Animal Health Research Unit, US Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, PO Box 952, Auburn, Alabama 36831-0952, USA
2Department of Pathobiology, College of Veterinary Medicine, Auburn University, Auburn, Alabama 36830, USA
3Department of Genetics, University of Alabama at Birmingham, Birmingham, Alabama 35294, USA

ABSTRACT: A multiplex PCR (m-PCR) method was developed for simultaneous detection of 3 important fish pathogens in warm water aquaculture. The m-PCR to amplify target DNA fragments from Flavobacterium columnare (504 bp), Edwardsiella ictaluri (407 bp) and Aeromonas hydrophila (209 bp) was optimized by adjustment of reaction buffers and a touchdown protocol. The lower detection limit for each of the 3 bacteria was 20 pg of nucleic acid template from each bacteria per m-PCR reaction mixture. The sensitivity threshold for detection of the 3 bacteria in tissues ranged between 3.4 × 102 and 2.5 × 105 cells g–1 of tissue (channel catfish Ictalurus punctatus Rafinesque). The diagnostic sensitivity and specificity of the m-PCR was evaluated with 10 representative isolates of each of the 3 bacteria and 11 other Gram-negative and 2 Gram-positive bacteria that are taxonomically related or ubiquitous in the aquatic environment. Except for a single species (A. salmonicida subsp. salmonicida), each set of primers specifically amplified the target DNA of the cognate species of bacteria. m-PCR was compared with bacteriological culture for identification of bacteria in experimentally infected fish. The m-PCR appears promising for the rapid, sensitive and simultaneous detection of Flavobacterium columnare, E. ictaluri and A. hydrophila in infected fish compared to the time-consuming traditional bacteriological culture techniques.

KEY WORDS: Multiplex-PCR · Fish · Edwardsiella · Flavobacterium · Aeromonas

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