Analysis of gyrB and toxR gene sequences of Vibrio hollisae and development of gyrB- and toxR-targeted PCR methods for isolation of V-hollisae from the environment and its identification

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using c onventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identifica tion of V. hollisae. The two genes are presumed to be conserved among the b acterial species (gyrB) or among the species of the genus Vibrio (toxR), A portion of the gyrB sequence of V. hollisae mas cloned by PCR using a set o f degenerate primers. The sequence showed 80% identity with the correspondi ng Vibrio parahaemolyticus gyrB sequence. The toxR gene of V. hollisae was cloned utilizing a htpG gene probe derived from the V. parahaemolyticus htp G gene, which is known to be linked to the toxR gene in V. hollisae. The co ding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybrid ization tests using the DNA probes derived from the two genes of V. hollisa e indicated that these gene sequences could be utilized for differentiation of V. hollisae from other Vibrio species and from microorganisms found in marine fish, PCR methods targeting the two gene sequences were established, Both PCR methods were shown to specifically detect the respective target s equences of V. hollisae but not other organisms. A strain of V. hollisae ad ded at a concentration of 1 to 10(2) CFU/ml to alkaline peptone water conta ining a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37 degrees C followed by quick DNA extraction wit h an extraction kit and 35-cycle PCR specific for the Ti. hollisae toxR gen e, We conclude that screening of seafood samples by this 35-cycle, V. holli sae toxR-specific PCR followed by isolation on a differential medium and id entification by the above htpG- and toxR-targeted PCR methods, can be usefu l for isolation from the environment and identification of V. hollisae.