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Vibrio cholerae identification based on molecular sequence data has been hampered by a lack of sequence variation from the closely relatedVibrio mimicus . The two species share many genes coding for proteins, such asctxAB , and show almost identical 16S DNA coding for rRNA (rDNA) sequences. Primers targeting conserved sequences flanking the 3′ end of the 16S and the 5′ end of the 23S rDNAs were used to amplify the 16S-23S rRNA intergenic spacer regions ofV. cholerae andV. mimicus . Two major (ca. 580 and 500 bp) and one minor (ca. 750 bp) amplicons were consistently generated for both species, and their sequences were determined. The largest fragment contains three tRNA genes (tDNAs) coding for tRNAGlu , tRNALys , and tRNAVal , which has not previously been found in bacteria examined to date. The 580-bp amplicon contained tDNAIle and tDNAAla , whereas the 500-bp fragment had single tDNA coding either tRNAGlu or tRNAAla . Little variation, i.e., 0 to 0.4%, was found amongV. cholerae O1 classical, O1 El Tor, and O139 epidemic strains. Slightly more variation was found against the non-O1/non-O139 serotypes (ca. 1% difference) andV. mimicus (2 to 3% difference). A pair of oligonucleotide primers were designed, based on the region differentiating all ofV. cholerae strains fromV. mimicus . The PCR system developed was subsequently evaluated by using representatives ofV. cholerae from environmental and clinical sources, and of other taxa, includingV. mimicus . This study provides the first molecular tool for identifying the speciesV. cholerae .

Analysis of 16S-23S rRNA Intergenic Spacer Regions of Vibrio cholerae and Vibrio mimicus