Vibrio alginolyticus gyrB sequence analysis and gyrB-targeted PCR identification in environmental isolates

gyrB fragments (about 1.2 kb) of 9 Vibrio alginolyticus strains were sequenced, and their phylogenetic relationship with other closely related Vibrio species was analyzed. All the V alginolyticus strains grouped into one strongly supported cluster in the phylogenetic tree. There were 54 base variations among the 1167 bp mutual gyrB regions of 11 V alginolyticus strains; all the V alginolyticus strains shared the same amino acid sequence except V alginolyticus ATCC 17749. Based on the gyrB sequences, we designed 2 primers for specific PCR identification of V alginolyticus. Fifty-two bacterial strains from 12 genera were used to test the PCR specificity, and only V alginolyticus strains produced the predicted 568 bp amplification fragment. In addition, PCR screening of 50 randomly selected environmental strains, grown on thiosulfate citrate bile salts-sucrose (TCBS) medium, gave rise to a positive amplification result for V alginolyticus from 37 of them. To further confirm accuracy of PCR identification, biochemical identification of the 50 strains was carried out. Strains giving positive PCR amplification were biochemically identified as V alginolyticus, while strains that gave negative results were biochemically identified as other Vibrio or non-Vibrio species. Using the basic local alignment search tool (BLAST), gyrB sequences obtained from 2 randomly selected strains (YJ0666 and YJ16713) of the 37 PCR-positive strains showed highest identity values with V. alginolyticus strains (>96%). Thus, our results demonstrated that gyrB is a good marker for molecular identification of V alginolyticus, and a gyrB-based PCR method was successfully developed.