Study On The Optimization And Application Of Rapid Enrichment Methods For Rapid Detection Of Pathogenic Microorganisms

Pathogenic microorganisms have become the No.1 killer of human health,of which most are food-borne pathogens.The food poisoning and deaths caused by food-borne pathogens have frequently occurred in the world,the research on the technology of food-borne pathogenic bacteria detection has become a hot spot for international scholars,and the focus of the research is on rapid detection technology.However,in the rapid detection,especially molecular biology techniques,pre-enrichment process is required,how to shorten the pre-enrichment time and improve the enrichment effect is a problem to solve that in the development of rapid detection.In this paper,several common foodborne pathogens were studied and the capsaicin soybean broth(TSB)was used as the basic medium,adding inoculum and bacteriostatic agent to it,single factor optimization experiment and response surface optimization experiment were used to determine a kind of enrichment medium which can enrich 4 common foodborne pathogens simultaneously,using LAMP kit to detect infected fruits and vegetables,to determine the application of bacteria culture effect.The main results are as follows:Through the study of the main components in the liquid culture medium of different strains,and analyzing the function of the constituents,the capsaicin soybean broth(TSB)was used as the basic medium,and selection of mannitol and sodium pyruvate as increasing agent,lithium chloride and bile salt as antibacterial agent,to determine the culturing formula composition of the culture medium.Optimized by single factor test by main effect in bacterial solution on the basis of factors,and at the same time,the addition amount of the bacteria and the antibacterial agent was optimized to determine the amount of each component in the culture broth:sodium chloride 10 g/L,mannitol 2 g/L,pyruvate 3 g/L,lithium chloride 1 g/L,bile salt 0.1 g/L.In order to optimize the enrichment medium,Design-expert software was used to design the 5 factors and 3 levels experiments.Through the contour map and 3D image analysis,the final formula of the culture medium was determined:tryptone 17 g/L,peptone 3 g/L,two g/L,2.5 sodium hydrogen phosphate and sodium chloride 10.5 g/L,glucose 2.5 g/L and 2 g/L mannitol,sodium pyruvate,3 g/L lithium chloride 0.8 g/L,bile salt 0.08 g/L.The influencing factors of 4 kinds of pathogen response as follows:Salmonella,bile salt>lithium chloride>sodium>chloride>sodium pyruvate>mannitol;Staphylococcus aureus,bile salt>lithium chloride>sodium chloride>sodium pyruvate>mannitol;Listeria monocytogenes,bile salt>lithium chloride>chloride sodium>mannitol>sodium pyruvate;Escherichia coli 0157,bile salt>lithium chloride>sodium chloride>sodium pyruvate>mannitol.With infection of fruits and vegetables experiments,and through the LAMP kit detection,and GB method re-examination,the results show that,broth have a good enrichment of Salmonella and Escherichia coli O157 bacteria,the detection rate in fruits and vegetables was 100%;the detection rate in vegetables of Listeria monocytogenes was 100%,while the detection rate in fruits was up than 90%;the detection rate in melon fruit of Staphylococcus aureu was low,the rests can be detected in other fruits and vegetables.The enrichment medium can be combined with artificial inoculation of fruits and vegetables,and the detection rate is high.