Analysis of gyrB and toxRGene Sequences of Vibrio hollisae and Development ofgyrB- and toxR-Targeted PCR Methods for Isolation of V. hollisae from the Environment and Its Identification

摘要

Isolation of Vibrio hollisae strains, particularly from the environment, is rare. This may be due, in part, to the difficulty encountered when using conventional biochemical tests to identify the microorganism. In this study, we evaluated whether two particular genes may be useful for the identification of V. hollisae. The two genes are presumed to be conserved among the bacterial species (gyrB) or among the species of the genus Vibrio(toxR). A portion of the gyrB sequence ofV. hollisae was cloned by PCR using a set of degenerate primers. The sequence showed 80% identity with the correspondingVibrio parahaemolyticus gyrB sequence. The toxRgene of V. hollisae was cloned utilizing a htpGgene probe derived from the V. parahaemolyticus htpG gene, which is known to be linked to the toxR gene in V. hollisae. The coding sequence of the cloned V. hollisae toxR gene had 59% identity with the V. parahaemolyticus toxR coding sequence. The results of DNA colony hybridization tests using the DNA probes derived from the two genes of V. hollisae indicated that these gene sequences could be utilized for differentiation of V. hollisae from otherVibrio 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 sequences of V. hollisae but not other organisms. A strain of V. hollisae added at a concentration of 1 to 102 CFU/ml to alkaline peptone water containing a seafood sample could be detected by a 4-h enrichment incubation in alkaline peptone water at 37°C followed by quick DNA extraction with an extraction kit and 35-cycle PCR specific for the V. hollisae toxR gene. We conclude that screening of seafood samples by this 35-cycle, V. hollisae toxR-specific PCR, followed by isolation on a differential medium and identification by the abovehtpG- and toxR-targeted PCR methods, can be useful for isolation from the environment and identification ofV. hollisae.