DAO 56:207-214 (2003)  -  doi:10.3354/dao056207

Genotyping of Flavobacterium psychrophilum using PCR-RFLP analysis

Shotaro Izumi1,2,4,*, Futoshi Aranishi2, Hisatsugu Wakabayashi1,3

1Department of Aquatic Bioscience, Graduate School of Agricultural and Life Sciences, The University of Tokyo, Yayoi, Bunkyo, Tokyo 113-8657, Japan
2Molecular Biology Division, National Institute of Fisheries Science, Fukuura, Kanazawa, Yokohama 236-8648, Japan
3HW Fish Health Laboratory, Sendagi, Bunkyo, Tokyo 113-0022, Japan
4Present address: Molecular Biology Division, National Institute of Fisheries Science, Fukuura, Kanazawa, Yokohama 236-8648, Japan

ABSTRACT: Genetic variability among 242 strains of Flavobacterium psychrophilum was characterized using polymerase chain reaction (PCR) followed by restriction fragment length polymorphism (RFLP) analysis. Universal Primers GYR-1 and GYR-1R, which were designed to amplify the gyrase subunit B gene (gyrB), yielded a 1178 bp PCR product encoding gyrB and a 290 bp PCR product of anonymous DNA from all F. psychrophilum strains tested. In the RFLP analysis of the anonymous 290 bp DNA marker, the restriction enzyme HinfI generated 2 cleavage patterns (Genotypes A and B). Genotype A was found only in isolates from ayu (n = 109), while Genotype B was found in isolates from coho salmon (n = 11), ayu (n = 35), rainbow trout (n = 43) and other fishes (n = 44). In the second experiment, Primers PSY-G1F and PSY-G1R specific for F. psychrophilum, were used to amplify gyrB. The specific primer pair amplified the expected size (1017 bp) PCR product from all F. psychrophilum strains. In the RFLP analysis of the gyrB, the restriction enzyme RsaI produced 2 genotypes, R and S. Genotype R was found in isolates from coho salmon (n = 6), ayu (n = 27), rainbow trout (n = 39) and other fishes (n = 4). Genotype S was found in isolates from coho salmon (n = 5), ayu (n = 117), rainbow trout (n = 4) and other fishes (n = 40).


KEY WORDS: Genotyping · Flavobacterium psychrophilum · PCR-RFLP · Bacterial cold-water disease · gyrB gene


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