Effect Of Environmental Factors On The Growth And Rapid Detection Method Of Vibrio Parahaemolyticus

Vibrio parahaemolyticus?VP?is a halophilic,gram-negative bacterium that is widely distributed in saline environments,such as ocean and salt lake.Some kinds of VP are pathogen and can infect a variety of animals,lending to clinical symptoms,e.g.acute gastroenteritis.Recently,the number of food poisoning incidents caused by VP is rising rapidly.In other words,the presence of VP is a serious risk of the ecological environment,as well as human health.Therefore,the investigations of the effect of environmental factors on its growth and the rapid detection method are of great significance for not only scientific research but also human health,e.g.food safety.Since the 21^st century,significant attention has been paid to the effect of environmental factors?e.g.temperature,salinity,pH?on the growth of VP.However,nearly all the studies were based on the response of single factor.The interaction of these effects is not understood clearly because the lack of correlation analysis.To get insight into the relationship between the target bacterium and environmental factors,better analysis methodology,by which the law of combing function can be figured out,is still a necessary issue.Up to now,various methods,such as conventional culture,immunization,PCR,to name a few,have been developed for the detection of VP.In particular,loop-mediated isothermal amplification method?LAMP?,which exhibited charming properties of high specificity and efficiency,has also been used for the detection recently.However,almost all the performances are focused on the assay of aquatic products and food.Nobody has concerned its existing in the source environment.As well known,apart from the process,living environment is the single source of VP to contaminate the aquatic products.Therefore,to monitoring accurately its concentration in environmental samples is also meaningful for human activities.However,to the best of our knowledge,there is no report on it so far.In this thesis,there are three main sections.In the first section,the background,concept and goal of the research was reviewed.To figure out the law of combing function,in the second section the effects of temperature,salinity,pH,phosphate,nitrate and ammonium on the growth of VP were investigated with response surface methodology?RSM?.Firstly,Box-Behnken design was used to optimize the three critical internal factors of temperature,salinity and pH,using optical density(OD₆₀₀)as marker of the growth of VP.It was found that for these factors the optimum parameters were 34.5?,3%and pH 8.0.Then based on these results,the combing effect of phosphate,nitrate and ammonium on the growth of VP was further studied with the same method to set up a response model.The equation determining coefficient?R²?was 0.9217.The correction coefficient?R²?was 0.8211.The regression model?F?was 9.16?P=0.004?.These results indicated that the model proposed here had satisfactory accuracy.Results obtained from verified testing were in good agreement with those obtained with the model.Clearly,phosphate,nitrate and ammonium all exhibited negative effect on the growth of VP.These outcomes are beneficial to the culture of VP and even the early warnings of marine environment and sea food safety.In the third section a rapid method was constructed based on a visual LAMP technology to detect VP,which has highly conserved genes of tlh.Briefly,bacteria in seawater samples were collected with filtration;then the collected bacteria were used as reaction template to perform LAMP,without the step of DNA extraction.Positive or negative results were identified by the color change of hydroxy naphthol blue with naked eyes.Under the optimized conditions,the detection limit was 167 CFU/mL?1 h?.In comparison with traditional PCR methods,the visual LAMP proposed here has the advantage of distinguish efficiency.Including the consumption of sample pretreatment,2h is enough for the whole performance of detection.Meanwhile,its sensitivity is about10-fold higher than PCR's.The visual LAMP method was applied to detect V.parahaemolyticus in real seawater samples collected from Qingdao coastal area.The results were in good agreement with that obtained with classical culture method and PCR method.No involvement of bulky instrumentation,plus the rapid response and simple operation,it's reasonable to believe that the new method is promising for constructing new working platforms,by which efficient and on-site surveys of target marine microorganisms can be achieved.