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Real-Time PCR Viability Assay for Vibrio and Total Bacteria in Seafood and Water


The goal of this project is to develop multi-probes using our Ethidium Bromide Monoazide Real-time PCR assay (EMA-Multi Rti-PCR) for the simultaneous detection and quantification of three representative viable Vibrio spp.and a number of viable total bacteria and to provide rapid and cost-effective microbiological methods for the routine monitoring and risk assessment of seafood and marine water quality. <P> The specific objectives of this proposed 3-year project are: 1) to design the four sets of primers and probes which target conserved genes for total bacteria and specific genes for Vibrio spp. (V. vulnificus, V. parahaemolyticus, V. anguillarum); 2) to optimize the Multi Rti-PCR conditions and apply it to pure and mixed bacterial cultures for verification; 3) to develop a PCR-sampling method to eliminate PCR inhibitors from fish and shellfish tissue and optimize EMA treatment to enumerate the total number of viable bacteria and viable Vibrio spp. from bacteria-seeded fish fillets and oysters; 4) to detect the total number of viable bacteria and Vibrio spp. from the aquacultured and/or wild caught fish and shellfish and marine water by using the EMA-Multi Rti-PCR assay; 5) to evaluate the correlation between fish and shellfish quality and bacterial population in the marine aquaculture ecosystem.<P> This project is our first effort to enhance the research capacity in the Food Science Program and will provide preliminary data that will enhance studies regarding metagenomic approaches in seafood safety and marine ecosystems. This research is the first collaboration among food scientists, aquaculturists, biostatisticians and USDA-ARS scientists at Delaware State University will provide an integrated multidisciplinary approach toward developing a new methodology and solving an important agricultural problem. Students and faculty in this project are expected to prepare oral presentations at scientific meetings and to submit manuscripts in peer-reviewed journals.

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Non-Technical Summary: <BR>Microbial spoilage and fish and shellfish diseases cause significant economic loss to the industry and to marine aquaculture. Outbreaks of seafood-borne illness regularly occur in the US. In this project, Vibrio. vulnificus, V. parahaemolyticus, and V. anguillarum were selected as target pathogens that cause human and fish diseases. The purpose of this project is to develop a novel DNA amplification method (EMA-multi Rti-PCR) for the simultaneous detection and quantification of three representative viable Vibrio spp. and a number of viable total bacteria. This method will be applied to assessing seafood safety and marine water quality. In addition, the total bacterial populations derived from fishery products and aquaculture environments will be studied in this project. This project will help reduce the quality control assessment time from 3-5 days to within 2 hours. The assay developed in this project could change the way pathogens are detected and provide rapid and cost-effective microbiological methods for the routine monitoring and risk assessment of seafood and marine water quality. <P> Approach: <BR> Development of multi-probes using the EMA (Ethidium Bromide Monoazide) real-time (Rti) PCR assay based on DNA amplification will provide a useful tool for the quality control of fishery products. Multiplex Rti-PCR allows various target organisms to be detected in the same reaction by using spectrally distinct dye-labeled probes. In addition, the Rti-PCR technique, involving treatment of samples with the intercalating agent EMA, is able to detect DNA only from viable microorganism. This project has two methodological innovations. The first one is the 4 sets of probes and primers to target specific and non-specific genes. This has never been done before for the assessment of fish and marine water safety and quality. The second innovation is that the Multi-Rti-PCR using EMA has not yet been applied to seafood samples to detect multi-targeted viable bacteria. This project will provide a new approach for the rapid and simple detection of Vibrios and total bacteria. This project seeks to enhance faculty knowledge and the new Food Science Program's research capability in the rapidly growing field of food safety and quality. The EMA-Multi Rti-PCR assay in this project will also provide graduate students, undergraduate students and scientists with the opportunity to undertake advanced research using state-of-the-art molecular technologies. The project helps narrow the technical gap of graduate research between the 1890 and 1862 institutions and provides students with novel educational and career opportunities in food companies, biotechnology and pharmaceutical companies, and government and research laboratories. The results collected by the students and research staff will be evaluated for standardization and accuracy. The impact of the project on research and education will be evaluated by: 1) application of the developed methodology to assess fish and water quality and safety, 2) number of presentations at scientific meetings or in publications, 3) funds attracted from Federal and state agencies, and 4) number of post-doctoral scientists, research technicians, and graduate and undergraduate students recruited to work on this project.

Lee, Jung-Lim
Delaware State University
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