Development of 16S rRNA targeted PCR methods for the detection and differentiation of Vibrio vulnificus in marine environments

A PCR method for the detection and differentiation of Vibrio vulnificus str ains was developed as an alternative to culture methods by using combined p rimers directed against the variable regions of 16S rRNA. Primers designed from two variable regions of the Vibrionaceae 16S rRNA (corresponding to nu cleotide numbers 1006 to 1023 and 1278 to 1258 in Escherichia coli 16S rRNA ) was found to be species-specific for V. vulnificus by PCR. Additionally, tri-primer PCR of 16S rRNA was evaluated for the differentiation of V. vuln ificus strains. Although the third primer, which was derived from the varia ble region, positions 454 to 473, cannot discriminate V. vulnificus from ot her bacteria, it was used to avoid the detection of type B 16S rRNA of this organism in PCR. The resulting 825 bp fragment in the presence of the 273 bp fragment, which is specific to V. vulnificus, in tri-primer PCR clearly differentiated type A 16S rRNA strains from type B. Enumeration of V. vulni ficus in the samples of oyster and environmental samples was done by most p robable numbers (MPN) method of five preenrichment tubes of alkaline pepton e water supplemented with polymyxin B following up the confirmation of posi tive tubes by streaking the samples onto mCPC agar or by 16S rRNA gene ampl ification. Higher numbers of presumptive V. I vulnificus confirmed by selec tive media compared with those confirmed by PCR method in MPN method sugges ted that there would be some bacteria that cannot be discriminated from V. vulnificus on mCPC agar in environmental samples. In the biotyping of the V . vulnificus isolates in oyster samples, the majority of the strains (92.5% ) belonged to biotype 1. and 7.5% of the strains belonged to biotype 2. How ever, strains of 16S rRNA of V. vulnificus isolates in the marine environme nt determined by tri-primer PCR appeared to be 35% type A and 65% type B. T hese results implied that the marine environment can serve as reservoir of both V. vulnificus biotypes 1 and 2, and strains of 16S rRNA type B were mo re frequent than strains of type A in that environment. (C) 2001 Elsevier S cience B.V. All rights reserved.