Development of a Rapid Monitoring System for Detection of Pathogens in Marine Aquaculture

pathogens in marine aquaculture waters. Phase II research will look to improve sample concentration, cell lysis, and DNA purification techniques that were developed in Phase I. More specifically, optimization of the prototype lysis/elution fluid will be further developed for cell lysis using BSL-1 organisms, Vibrio harveyi, a surrogate for pathogenic Vibrio ssp. Techniques will be developed to overcome qPCR inhibition related to co-concentration of organic material from sea water sample, including prefilatration of sediment and humic acid removal. A prototype DNA purification kit will also be developed and optimized to facilitate DNA purification from lysed V. harveyi concentrated from experimentally infected sea water. The system will be tested in-house as V. harveyi will be spiked into one liter of sea water at 0.1 CFU/mL (low level) and 10 CFU/mL (high level), concentrated, lysed, and genomic DNA purified and analyzed by real-time quantitative Polymerase Chain Reaction. Finally, an independent evaluation of the complete prototype concentration, lysis, and purification system will be performed.