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

Academic Article

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. © Inter-Research 2007.
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    Digital Object Identifier (doi)

    Author List

  • Panangala VS; Shoemaker CA; Van Santen VL; Dybvig K; Klesius PH
  • Start Page

  • 199
  • End Page

  • 208
  • Volume

  • 74
  • Issue

  • 3