Multiplex primer-extension assay for identification of six pathogenic vibrios

A multiplex Primer-Extension Reaction (PER) assay, was specifically designed for the identification, of the major human pathogenic Vibrio species (V. cholerae, V. parahaemolyticus, V. vulnificus, V. mimicus, V. alginolyticus and V. fluvialis) in fishery products. The assay, directed towards the rpoA gene, was tested on a total of 287 samples representing six Vibrio species and ten non-Vibrio species. The primers used in the preliminary PCR, designed in well conserved regions upstream and downstream of the diagnosis sites, successfully amplified a 284 bp fragment. The diagnosis sites were simultaneously interrogated using a multiplex PER and the results were confirmed by fragment sequencing. The proposed test provides an appropriate tool to monitor the presence of these human pathogenic species in seafood samples and to evaluate the potential hazard for consumers.