Block 1. Development of simple molecular methods for detection and enumeration of (months 0-48).
Initially a review of both molecular and classical approaches for the enumeration and characterisation of pathogenic vibrios in seafood will be completed (partners 21.CEFAS.). Comparative performance assessment of selected molecular techniques versus classical methods will also be undertaken (partners 21.CEFAS, 23.ISS, 3.IFREMER). Where necessary molecular procedures will be developed for application within the project, in conjunction with the standardised conventional methods. It is possible that specific target nucleic acid sequences will not be identified for the pathogens or their virulence markers. If necessary, the conventional techniques will be used to supplement the developed molecular ones in order to ensure appropriate specificity.
Block 2. Development of real-time PCR procedures for Vibrio spp (months 10-48)
Real-time PCR procedures will be developed for the enumeration of the bacterial pathogens in seafoods. Methods will be established for both quantitation of pathogenic bacteria and total numbers (partner 3.IFREMER). The performance of these will be evaluated and validated using spiked and naturally contaminated samples (partners 23.ISS, 3.IFREMER, 44.USC).
Block 3. Depuration and survival studies on Vibrio spp and (months 10-48).
Developed techniques will be used to investigate depuration kinetics for Vibrio spp in shellfish and their survival in post-harvest seafood (partners 21.CEFAS, 23.ISS).
Block 4. Characterisation of Vibrio (months 10-48)
Development and application of molecular typing methods and genetic fingerprinting methods will be used to characterise the epidemiological significance of vibrio strains isolated from seafood. It is possible that insufficient isolates are available from field validation studies for strain characterisation. As a contingency, strains would be sought from culture collections of participants and other European institutes and supplemented with isolates from collaborating laboratories in Japan and the US. Standard methods will be produced (partners 21.CEFAS, 9.IFL) and circulated to the consortium laboratories (partners 21.CEFAS, 23.ISS, 3.IFREMER, 44.USC, 9.IFL). Working in collaboration with participant laboratories genomic fingerprints will be generated for strains isolated from the integrated European surveillance activity. These fingerprints will then be analysed and clonal relationships with significant pandemic/epidemic strains assessed. This will facilitate informed, appropriate assessments of the potential risks to the consumer.