Development of a PCR assay targeting the rpoA gene for the screening of Vibrio genus

A polymerase chain reaction (PCR) method based on the RNA polymerase alpha subunit (rpoA) gene was developed for the detection of the Vibrio genus. The specific primers were designed aligning the rpoA gene sequences available in GenBank of all Vibrio species. The specificity of the primers was tested against 35 Vibrio species. In addition, 12 species phylogenetically related to the Vibrio genus were used as negative control. Moreover, in order to eliminate any false-negative results, bacteriumspecific primers targeting the 16S rRNA gene were introduced in the test as a noncompetitive internal amplification control. The rpoA primers correctly amplified all the Vibrio species considered. No cross-reaction was observed when tested against closely related species. To estimate the applicability of this method, 336 Vibrio wildtype strains isolated from Italian aquaculture products and from imported seafoods were tested. The sensitivity, tested using serial dilutions of different pure cultures of certified strains, resulted to 103 colony-forming units per milliliter. The assay proved to be specific, rapid, and reliable. It can be proposed as a routine screening technique for the confirmation of Vibrio genus in isolated colonies.