title: Multiplex-PCR for simultaneous detection of 3 bacterial fish pathogens, Flavobacterium columnare, Edwardsiella ictaluri, and Aeromonas hydrophila.
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 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.
authors.affiliation: Aquatic Animal Health Research Unit, U.S. Department of Agriculture, Agricultural Research Service, Aquatic Animal Health Research Unit, P.O. Box 952, Auburn, Alabama 36831-0952, USA. vpanangala@ars.usda.gov
authors.forename: Victor S
authors.initials: VS
authors.lastname: Panangala
authors.forename: Craig A
authors.initials: CA
authors.lastname: Shoemaker
authors.forename: Vicky L
authors.initials: VL
authors.lastname: Van Santen
authors.forename: Kevin
authors.initials: K
authors.lastname: Dybvig
authors.forename: Phillip H
authors.initials: PH
authors.lastname: Klesius
mesh_terms: D016980:Aeromonas hydrophila / Q000302:isolation & purification*; D000818:Animals; D001427:Bacterial Toxins / Q000235:genetics; D001431:Bacteriological Techniques / Q000379:methods / Q000662:veterinary; D017931:DNA Primers / Q000737:chemistry; D021903:DNA, Ribosomal Spacer / Q000235:genetics; D020610:Edwardsiella ictaluri / Q000302:isolation & purification*; D005393:Fish Diseases / Q000175:diagnosis* / Q000382:microbiology; D005417:Flavobacterium / Q000302:isolation & purification*; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000662:veterinary*; D007059:Ictaluridae / Q000382:microbiology; D016133:Polymerase Chain Reaction / Q000662:veterinary; D052899:Pore Forming Cytotoxic Proteins / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012680:Sensitivity and Specificity; D014871:Water Microbiology
chemical_list: D001427:Bacterial Toxins; D017931:DNA Primers; D021903:DNA, Ribosomal Spacer; D052899:Pore Forming Cytotoxic Proteins; D012336:RNA, Ribosomal, 16S; C032676:aerolysin