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

• Published in: Diseases of aquatic organisms 2007-03, Vol.74 (3), p.199-208
• Main Authors: PANANGALA, Victor S, SHOEMAKER, Craig A, VAN SANTEN, Vicky L, DYBVIG, Kevin, KLESIUS, Phillip H
• Format: Article
• Language: English
• Subjects: Aeromonas hydrophila - isolation & purification; Animal aquaculture; Animal productions; Animals; Bacterial Toxins - genetics; Bacteriological Techniques - methods; Bacteriological Techniques - veterinary; Biological and medical sciences; DNA Primers - chemistry; DNA, Ribosomal Spacer - genetics; Edwardsiella ictaluri - isolation & purification; Fish Diseases - diagnosis; Fish Diseases - microbiology; Flavobacterium - isolation & purification; Fundamental and applied biological sciences. Psychology; Gram-Negative Bacterial Infections - diagnosis; Gram-Negative Bacterial Infections - veterinary; Ictaluridae - microbiology; Pisciculture; Polymerase Chain Reaction - veterinary; Pore Forming Cytotoxic Proteins - genetics; RNA, Ribosomal, 16S - genetics; Sensitivity and Specificity; Vertebrate aquaculture; Water Microbiology
• ISSN: 0177-5103; 1616-1580
• DOI: 10.3354/dao074199

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 x 10(2) and 2.5 x 10(5) 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.