Development of a polymerase chain reaction assay for identification and detection of the fish pathogen Flavobacterium psychrophilum.
A PCR-based method was developed to identify and detect Flavobacterium psychrophilum, the causative agent of "cold-water disease" and "rainbow trout fry syndrome" in salmonid fish. Two oligonucleotide primers were designed by comparing the 16S rRNA sequence of all taxa in the genus Flavobacterium and of representative species in most related genera within rRNA superfamily V. Purified chromosomal DNAs from all these bacterial species, from 25 F. psychrophilum isolates and from several other fish-pathogenic bacteria were used to assess the specificity of the reaction. Amplification products were generated only with F. psychrophilum DNA. The detection level, equivalent to approximately 10 to 100 bacterial cells, was increased 10-fold by hybridization with a radioactive probe. Preliminary experiments demonstrated that this procedure can also be applied to samples of infected tissue. This PCR assay is therefore a rapid, specific, and sensitive alternative to conventional plate culture methods for the identification and detection of F. psychrophilum.
Laboratoire de Microbiologie et Biochimie Appliquée, Ecole Nationale des Ingénieurs des Travaux Agricoles de Bordeaux, Gradignan, France.
M C
MC
Urdaci
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C
Chakroun
D
D
Faure
J F
JF
Bernardet
D000818:Animals; D015139:Blotting, Southern / Q000662:veterinary; D004269:DNA, Bacterial / Q000737:chemistry; D004587:Electrophoresis, Agar Gel / Q000662:veterinary; D005393:Fish Diseases / Q000175:diagnosis / Q000382:microbiology*; D005417:Flavobacterium / Q000737:chemistry / Q000276:immunology / Q000302:isolation & purification*; D005618:Fresh Water; D016905:Gram-Negative Bacterial Infections / Q000175:diagnosis / Q000382:microbiology / Q000662:veterinary*; D017686:Oncorhynchus mykiss / Q000382:microbiology*; D016133:Polymerase Chain Reaction / Q000379:methods / Q000662:veterinary*; D012313:RNA / Q000737:chemistry; D012329:RNA, Bacterial / Q000737:chemistry; D012336:RNA, Ribosomal, 16S / Q000737:chemistry; D012680:Sensitivity and Specificity; D012867:Skin / Q000382:microbiology; D013154:Spleen / Q000382:microbiology; D014871:Water Microbiology
D004269:DNA, Bacterial; C034477:RNA primers; D012329:RNA, Bacterial; D012336:RNA, Ribosomal, 16S; D012313:RNA