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Journal of Clinical Microbiology, January 2007, p. 134-140, Vol. 45, No. 1
0095-1137/07/$08.00+0     doi:10.1128/JCM.01544-06
Copyright © 2007, American Society for Microbiology. All Rights Reserved.

Identification of Vibrio Isolates by a Multiplex PCR Assay and rpoB Sequence Determination

Foodborne and Diarrheal Diseases Branch, Division of Bacterial and Mycotic Diseases, National Center for Infectious Diseases, Centers for Disease Control and Prevention, Atlanta, Georgia 30333,¹ Battelle Memorial Institute, Battelle Science and Technology International, Chemical and Environmental Technologies, Atlanta Analytical Chemistry Group, 2971 Flowers Road South, Atlanta, Georgia 30341²

Received 25 July 2006/ Returned for modification 1 September 2006/ Accepted 2 November 2006

Vibrio, a diverse genus of aquatic bacteria, currently includes 72 species, 12 of which occur in human clinical samples. Of these 12, three species—Vibrio cholerae, Vibrio parahaemolyticus, and Vibrio vulnificus—account for the majority of Vibrio infections in humans. Rapid and accurate identification of Vibrio species has been problematic because phenotypic characteristics are variable within species and biochemical identification requires 2 or more days to complete. To facilitate the identification of human-pathogenic species, we developed a multiplex PCR that uses species-specific primers to amplify gene regions in four species (V. cholerae, V. parahaemolyticus, V. vulnificus, and V. mimicus). The assay was tested on a sample of 309 Vibrio isolates representing 26 named species (including 12 human pathogens) that had been characterized by biochemical methods. A total of 190 isolates that had been identified as one of the four target species all yielded results consistent with the previous classification. The assay identified an additional four V. parahaemolyticus isolates among the other 119 isolates. Sequence analysis based on rpoB was used to validate the multiplex results for these four isolates, and all clustered with other V. parahaemolyticus sequences. The rpoB sequences for 12 of 15 previously unidentified isolates clustered with other Vibrio species in a phylogenetic analysis, and three isolates appeared to represent unnamed Vibrio species. The PCR assay provides a simple, rapid, and reliable tool for identification of the major Vibrio pathogens in clinical samples, and rpoB sequencing provides an additional identification tool for other species in the genus Vibrio.

Published ahead of print on 8 November 2006.