Purpose: The target was to design a real-time PCR assay that discriminates 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 a 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: With novel targets, false-positive and false-negative results, known from other methods using targets like tlh or hlyA, were 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 was superior to other Vibrio detection methods. There were no false positive results for all 73 tested samples of 54 closely-related species and bacteria from the same habitat. The sensitivity of the foodproof® Vibrio Detection LyoKit is one genomic equivalent (GE)/reaction for species detection and 10 – 25 GE/reaction for toxin detection. The assay is compatible with all 21 tested raw and processed seafood matrices, such as 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: This assay met the demands of testing laboratories by demonstrating 100% specificity and high sensitivity. 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.