Rapid Detection Of Escherichia Coli O157:H7 And Salmonella In Shrimp And Mechanism Of Cold Tolerance

Food-borne pathogens are diverse in nature and these pathogens enter food through various media,causing food-borne illnesses and have become an important public health issue that threatens health of people.Understanding the characteristics of food-borne pathogens and performing rapid and effective testing of them is the key to prevent and control food-borne diseases.In this study,Escherichia coli O157:H7 and Salmonella were taken as the research object,and a rapid detection method using shrimp as a food matrix was studied from a technical perspective.At the same time,the mechanism of cold stress resistance of E.coli O157:H7 was discussed theoretically.The main research contents and results are as follows:The immunomagnetic separation systems of E.coli O157:H7 and Salmonella were constructed and optimized respectively.The magnetic beads were coated with the E.coli O157:H7 monoclonal antibody and the Salmonella polyclonal antibody,respectively.The optimal amount of the antibody was determined to prepare the immunomagnetic beads.Meanwhile,the amount of immunomagnetic beads added during the enrichment of the target bacteria by the immunomagnetic bead and the reaction time were also optimized.The results showed that the optimal amount of antibody was 50?g for E.coli O157:H7 immunomagnetic bead separation system and same to Salmonella immunomagnetic bead separation system,and the optimal amount of immunomagnetic beads was 80?L and 100?L,respectively.30min was the best reaction time for both E.coli O157:H7 and Salmonella immunomagnetic bead separation systems.The optimized separation systems had good stability.For 10~4CFU/mL target strains,the capture rate of E.coli O157:H7 immunomagnetic beads was 92.4%and the capture rates of Salmonella immunemagnetic beads for four common Salmonella strains were all over 74%.Two optimized immunomagnetic bead separation systems combined with Real time PCR were used to establish IMS-Real time PCR methods for the detection of E.coli O157:H7 and Salmonella in shrimp.The results showed that the methods had good specificity and stability,and direct detection time was about 3 h.The detection limit for E.coli O157:H7 was 5×10~1CFU/25 g,and the detection limits for four strains of Salmonella were slightly different,but still maintained at 10~3 CFU/25 g level.The corresponding detection limit of 10~0 CFU/25 g could be achieved by enriching the sample for detecting E.coli O157:H7 for 3 h and enriching the sample for detecting Salmonella by 5 h.Compared with the national standard assay to verify the proposed methods are fast and accurate.The transcriptional information of cold stressed E.coli O157:H7 and normal E.coli O157:H7 was compared by RNA-seq sequencing to reveal its cold tolerance mechanism.Through sequencing,1254 significant differentially expressed genes were detected under cold stress,and 425 important related genes were screened out after analysis.These genes mainly involved in carbohydrate metabolism,amino acid metabolism,energy metabolism,lipid metabolism,and biosynthesis of other secondary metabolites.Among these differentially expressed genes,the rpoS gene was significantly up-regulated,which may be the key switch in the regulation of cold stress resistance of E.coli O157:H7.The cold shock protein genes were mainly expressed in the acclimation stage at the early stage of cold stress,and not a key factor for the long-term survival of the bacteria at low temperature.In the cold stress group,the membrane-associated genes lpxP,yohJ,nlpD,bolA,and the genes livH,ilvE,livM,livG,tesB and fadB,which involved in the synthesis of branched chain amino acids and unsaturated fatty acids all showed an upward trend.It indicated that when E.coli O157:H7 was under cold stress,the fluidity of the cell membrane could be maintained by increasing the proportion of unsaturated fatty acids and methyl branches and cis fatty acids.At the same time,the expression of genes such as betA and betB,otsA and otsB were up-regulated,which indicated that E.coli O157:H7 accumulated betaine and synthesize trehalose to maintain osmotic balance,promote growth and metabolism,and provide cryogenic protection for cells.In summary,the established IMS-Real time PCR detection methods for E.coli O157:H7and Salmonella in shrimps are rapid,accurate,sensitive,and stable,providing technical means for the surveillance of pathogenic bacteria in aquatic products.The analysis of the transcriptional information of E.coli O157:H7 underlined its systematic mechanism of cold stress tolerance,which was helpful to the effective risk assessment of microbial safety during food processing,transportation and storage.And that also contributed to the establishment of more effective control technology based on its molecular mechanism.