Graduate School of Fisheries Sciences, Hokkaido University, Hakodate, Hokkaido 041-0821, Japan
The causative bacteria, Vibrio spp., have been isolated from the diseased sea urchin Strongylocentrotus intermedius at low water temperatures. To develop a specific identification method for the pathogens, species-specific nucleotide sequences in the 16S rRNA of Vibrio spp., were determined. We designed two PCR primers, DA2F and SK1F, based on the nucleotide sequences: DA2F forward primer (nucleotide numbers 456 to 476 in Escherichia coli 16S rRNA) for Date isolates and Sk1F forward primer (nucleotide numbers 455 to 478 in E. coli 16S rRNA) for Shiriuchi and Shikabe isolates.
The PCR product with about 1kbp was obtained in all causative bacteria from Date, Shiriuchi, Shikabe Breeding Centers and some of Vibrio reference strains by amplification with DA2F-1540R primer set, however, those from three Breeding Centers differed from the reference strains in the case of growth at 30°C. PCR amplification with SK1F-1540R primer set was able to differentiate the causative bacteria from Date and those from Shiriuchi and Shikabe. These results indicate that PCR amplification with two primers is useful for identification of Vibrio spp., which are the pathogens of cultured sea urchin.