Real-time PCR and nucleic acid hybridisation assays for the detection of total and thermostable direct and direct related haemolysins of V. parahaemolyticus have been developed. These procedures are based on the species-specific marker toxR. This was found to be the most suitable marker for differentiating V. parahaemolyticus from other Vibrio spp in European seafood. The hybridisation method is suitable for direct enumeration and allows detection and enumeration of potentially pathogenic strains which may provide early indication of the risk to the consumer from seafoods. The next step is to undertake an initial validation exercise involving all partner laboratories with a view to finalising procedures which will be presented to ISO SC9 and CEN WG6 in spring 2008. The real time PCR method again using primers and probes for the detection of the tdh and trh genes has been designed and optimised in seeded seawater. However further optimisation of DNA extraction procedures is required for bivalve shellfish matrices.
A standard operating procedure for a pulse field gel electrophoresis method for vibrios has also been produced. This has allowed characterisation of isolates indicating a high level of genetic diversity amongst seafood derived strains but which is not observed in clinical isolates.
