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

摘要

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 clusterin 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. Inaddition, 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 YJ167B) 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.