Separation Of Pathogenic Strains Of Vibrio Parahaemolyticus From Shellfish By LAMP And Its Growth Model Establish

Vibrio parahaemolyticus is a gram- negative bacillus which widely distributed along the offshore sea water and sediment, as well as in all kinds of seafood. It is also the current primary food-borne pathogens in china. Eating seafood contaminated with Vibrio parahaemolyticus can cause nausea, vomiting and other symptoms of acute gastroenteritis, especially the consumption of raw or half raw shellfish products such as oysters, can cause food poisoning frequently.From 1992 to 2013, Vibrio parahaemolyticus related statistics data, seafood in all major cities in C hina detection rate of Vibrio parahaemolyticus were over 30%, the highest detection rate can reach 80%, especially has a relatively high detection rate in shellfish and in the season of summer and autumn.Vibrio parahaemolyticu s has two kinds including pathogenic and non-pathogenic, and the majority Vibrio parahaemolyticus are non-pathogenic, However, the pollution survey data has not been the vice of virulence genes of pathogenic for further analysis and research, the test sample containing a non-pathogenic Vibrio parahaemolyticus. So it is hard to get to know the real situation through the detection rate.In order to make a further analysis of Vibrio parahaemolyticus carrying toxic gene in seafood, and get more real grasp of the risk. This study was to detect the pathogenic Vibrio parahaemolyticus in shellfish products based on loop- mediated isothermal amplification(LAMP), and set up its growth model use the isolated strains from shellfish, So as to provide data support to red uce Vibrio parahaemolyticus foodborne disease caused by eating shellfish, try to avoid or reduce the occurrence of food poisoning caused by Vibrio parahaemolyticus.In this study, 180 samples of 6 species commercial shellfish were purchased from shanghai local seafood market and detected on tdh and trh virulence strains of Vibrio paraem Lyticus by using the LAMP assay during August to October. Detection of PCR assay was compared with LAMP assay simultaneously. The results indicated that the detection rate of tdh and trh in commercial shellfish was 12.77 % and 11.66 % respectively, which were higher than the result detected by PCR with 10.55% and 7.77%. 2 strains of O3: K6 serotype Vibrio parahemolyticus were confirmed in 5 isolation virulence strains. But it was different in their PFGE stripe between the two O3: K6 strains. In conclusion, commerc ial shellfish was contaminated by virulence strains of Vibrio paraem Lyticus in shanghai local seafood market. Consumers should pay more attention to the potential threats of Vibrio paraem Lyticus in shellfish. The results also indicated that the LAMP method was suitable for the detection of virulence strains of Vibrio parahemolyticus in shel fish.Draw the growth curve of the separation Vibrio parahaemolyticus pathogenic strains, non-pathogenic and laboratory standard strains(pathogenic) in 35℃, 15℃ and 25℃, 5℃ in alkaline peptone water, oyster meat, and live oysters, and analysis with the data by using Excel and Curve Expert 1.4 software, in order to fit the data, Richards model, the MMF model, Gompertz model and Logistic model were used respectively, establish the most appropriated model for Vibrio parahaemolyticus pathogenic strains in shellfish products, after that make a conduct of the model validation. The experimental results show that:(1)In 3% Nacl alkaline peptone water non-pathogenic strains has higher growth rate than pathogenic strains in 35℃, 25℃ and 15℃, and have a minimum reduce speed in 5℃, that indicate non-pathogenic strains was more adaptable in growing environment of liquid matrix than pathogenic strains. In oyster meat, and live oysters, in a laboratory sample isolates standard strains had higher growth rate than laboratory standard strains(pathogenic) in 35℃, 25℃ and 15℃, 15℃ when sample deputy hemolytic separation strains of pathogenic vibrio bacteria with the highest level, minimum cut rate when it is 5℃, explain deputy hemolytic pathogenic vibrio than deputy hemolytic pathogenic vibrio more adapt to growth in the meat and living shel fish.(2)MMF model the best fitting effect in water matrix in 35℃, 25℃ and 15℃ and also the best fitting effect in living shellfish meat in 25℃ and 15℃. Richards model has the best fitting effect in shellfish meat matrix 35℃ in 35℃, 25℃ and the shellfish in vivo. Logistic model is suitable for the water, shellfish meat and living shellfish in 5℃, and also the optimal growth model in shellfish meat in15℃.The credibility of the temperature growth model were evaluated in live shellfish under laboratory conditions, maximum specific growth rate of residual are within the range of ± 0.05, this indicates that the prediction model is credible in 95% of the level.