Rapid Detection Techniques And Molecular Predictive Model Of Common Foodborne Pathogens In Litopenaeus Vannamei

Litopenaeus vannamei is one of the most important aquatic products in China and rest of Asia, as well as one of the most popular foods in these areas because of the delicacy and nutrition. Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important hazards in seafood products. Microbiological risk assessment was regarded as an important approach to effectively control these hazards. High-throughput detection techniques and efficient predictive model could provide powerful tool for assessing and control the health risk associated with foodborne pathogens in seafood products. Therofore, this study aimed to(1) develop a rapid, accurate and internationally accepted method for detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. in raw shrimp(Litopenaeus vannamei);(2) develop a rapid, accurate and efficient methods for simultaneous quantification of viable Vibrio parahaemolyticus, Listeria monocytogenes in raw shrimp(Litopenaeus vannamei);(3) develop and validate a molecular predictive model to describe the fate of naturally-occurring V. parahaemolyticus in raw shrimp(Litopenaeus vannamei) harvest from the major shrimp farm of Shanghai storage at different temperature(4, 7, 15, 20, 25 and 30 °C). The main content of this study were summarized as follows: 1. Development of a multiplex real-time PCR method for simultaneous detection of Vibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. in raw shrimpVibrio parahaemolyticus, Listeria monocytogenes and Salmonella spp. are important pathogens contaminating seafood in China. In this study, we developed an efficient multiplex real-time PCR for the simultaneous detection of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in raw shrimp without a prior enrichment step. In a test using 28 target and non-target strains only the targets were detected and two calibration curves, for pure cultures and artificially contaminated samples, were used to evaluate the efficiencies of this method. Amplification efficiencies of this multiplex real-time PCR were excellent in pure cultures and artificially contaminated shrimps. The limits of detection in artificially contaminated shrimps were 112 CFU/g for V. parahaemolyticus, 158 CFU/g for L. monocytogenes and 103 CFU/g for Salmonella spp. We validated this multiplex real-time PCR method on 48 commercial samples and the results were comparable to standard culture methods. This efficient multiplex real-time PCR, where each test takes only 50 minutes after DNA extraction, is a useful tool for high-throughput surveillance of V. parahaemolyticus, L. monocytogenes and Salmonella spp. in seafood products. 2. Quantifying viable Vibrio parahaemolyticus and Listeria monocytogenes simultaneously in raw shrimpA novel Taq Man-based multiplex real-time PCR method combined with propidium monoazide treatment was firstly developed for the simultaneous quantification of viable Vibrio parahaemolyticus and Listeria monocytogenes in raw shrimp. The optimization of PMA concentration showed that 100 μM was considered optimal to effectively inhibit 108 CFU/m L dead cells of both bacteria. The high specificity of primers and probes were confirmed on tests using 96 target and non-target strains. The optimized assay could detect as low as 101-102 CFU/g of each strain on the artificially contaminated shrimp, and its amplification efficiencies were up to 100% and 106% for V. parahaemolyticus and L. monocytogenes respectively. Furthermore this assay has been successfully applied to describe the behavior of these two pathogens in raw shrimps stored at 4°C. In conclusion, this PMA Taq Man-based multiplex real-time PCR technique, where the whole procedure takes less than 5 hours, provides an effective and rapid tool for monitoring contamination of viable V. parahaemolyticus and L. monocytogenes in seafood, improving seafood safety and protecting public health. 3. Development and validation of a molecular predictive model for the behavior of naturally-occuring Vibrio parahaemolyticus in post-harvest shrimpIn this study, we developed a molecular predictive model to describe the behavior of naturally-occurring V. parahaemolyticus in raw shrimp(Litopenaeus vannamei) harvest from the major shrimp farm of Shanghai storage at different temperature(4, 7, 15, 20, 25 and 30 °C). The concentration of naturally-occurring V. parahaemolyticus in raw shrimp was quantified by a real-time PCR method combined with propidium monoazide treatment. The Baranyi-model was applied to fit the V. parahaemolyticus growth and survival data. The Secondary model was produced using Square root model and Non-linear Arrhenius model. This model was validated by using the independent data obtained from raw shrimp harvested from the same region stored at 18, 23, 28 °C. Naturally-occurring V. parahaemolyticus inactivated at 4 and 7 °C with average rates of-0.019 and-0.025 Log CFU/g/h respectively. The average μmax at 15, 20, 25, and 30 °C were 0.044, 0.105, 0.179 and 0.336 Log CFU/g/h, and the average λ at 15, 20, 25, and 30 °C were 15.451, 7.259, 4.427 and 3.741 h respectively. The bias and accuracy factors of the secondary model for model-independent data were 0.963 and 1.066 for Square root model and 1.054 and 0.974 for Non-linear Arrhenius model. That indicated that the models could reliably predict the behavior of naturally-occurring V. parahaemolyticus in raw shrimp. Furthermore the behavior of naturally-occurring V. parahaemolyticus in post-harvest shrimp was significantly different from artificially contaminated V. parahaemolyticus in cooked shrimp. That indicated the traditional predictive model based on artificially contaminated V. parahaemolyticus may incorrectly estimate the risk of this pathogen. This molecular predictive model will be valuable for studying on the behavior of naturally-occurring V. parahaemolyticus and provide trustworthy tool for risk assessment of this important pathogen.