Application of a novel pathogenicity marker in a multiplex real-time PCR method to assess total and pathogenic Vibrio vulnificus in food and environmental samples

Pathogenic species of Vibrio genus, including Vibrio vulnificus, constitute a great challenge for food control agencies and a threat for consumers. V. vulnificus can appear in bivalve mollusks such as oysters, clams, and mussels. In addition, water, sediment, and plankton, have been described as reservoirs for this pathogen which constitutes the leading cause of death by consuming seafood in the United States.The aim of this study was to develop and pre-validate a rapid and reliable multiplex real-time PCR (qPCR) method for total and pathogenic V. vulnificus detection. Peptone, Sodium Chloride, Cellobiose (PNC) broth, with and without Colistin (PNCC) was evaluated according to international methods (ISO). The capacity of these broths to recover low numbers of pathogenic V. vulnificus in the presence of high numbers of interfering microorganisms was assessed. Finally, were used for food and environmental samples enrichment.In addition three different DNA extraction protocols were compared, but one of them proved to be better than the others regarding DNA concentration and purity obtained, and also regarding Ct values and final fluorescence obtained by qPCR.A qPCR efficiency above 90% was obtained, covering five orders of magnitude. The complete method achieved low limit of detection (3 cfu/25 g). All quality parameters of the method (relative sensitivity, specificity, and accuracy) returned values over 90% after analyzing 45 spiked samples. These results were obtained for all the targets analyzed (vvhA, vcgC and pilF).In this study the complete qPCR method developed was applied to 28 natural samples including a wide variety of seafood types and environmental samples (water), but no positive samples were detected for either target.