Purpose: The target was to design a real-time PCR assay, that can discriminate between V. parahaemolyticus, V. vulnificus and V. cholerae, and simultaneously detects and individually identifies the pathogenicity factors ctx, tdh, trh1 and trh2 by melting curve analysis in just one single reaction using sequence specific 5`Nuclease-probes. For convenience, the assay must be lyophilized.
Methods: Specificity (inclusivity/exclusivity) was tested with DNA extracts. Sample matrix compatibility, sensitivity and viability PCR were tested with genomic DNA and spiked samples.
Results: By using novel targets, false-positive and false-negative results, known from other methods using targets like, e.g., tlh or hlyA, are avoided. A total of 149 strains (74 V. parahaemolyticus, 26 V. vulnificus and 49 V. cholerae) were tested for inclusivity: With 100% specificity (inclusivity/exclusivity) for the detection of species and pathogenicity factors, the assay is superior to other methods for Vibrio detection. There were no false-positive results for all 73 tested samples of 54 closely related species and bacteria of the same habitat. The sensitivity of the foodproof® Vibrio Detection LyoKit is 1 genomic equivalent (GE) per reaction for species detection and 10 – 25 GE per reaction for toxin detection. The assay is compatible with all 21 tested raw and processed seafood matrices like whole squid, raw oysters or smoked salmon. The sample preparation includes a live/dead discrimination by using Reagent D, which efficiently removes DNA of at least 103 CFU/ml dead Vibrio.
Significance: One hundred percent specificity and high sensitivity meets the demands of testing laboratories. As seafood often is contaminated with dead cells of Vibrio spp., Reagent D treatment prevents false-positive results which may be encountered with other PCR methods.