| Bacteria can not grow on conventional solid media to form colonies or form tiny colonies invisible to the naked eye,but they still have life activities and are capable of being metabolized.This state of survival is referred to as the viable but non-culturable state(VBNC state).In the routine monitoring of foodborne pathogens,the VBNC state bacteria cannot form colonies on solid media,and thus,the traditional coating plate detection method cannot detect VBNC cells,which is easy to cause missed detection of VBNC cells.In recent years,frozen food industry has also been rapid development and becomes one of the important ways of traditional food industrialization in China.The experimental system of this topic is frozen beef meatballs in quick-frozen food,because the beef pills are rich in protein,amino acids and other nutrients,which are suitable for the growth and reproduction of foodborne pathogens.Enterohemorrhagic Escherichia coli(EHEC)is a subtype of Escherichia coli,E.coli O157:H7 is one of the main serotype EHEC,which is one of the most important food borne pathogens,can cause symptoms of intestinal disease and fatal diseases including hemorrhagic colitis,hemolytic anemia and hemolysis hemolytic uremic syndrome.And E.coli O157:H7 is the most common food borne pathogen in beef food.In addition,the factors such as preservatives and low temperature are easy to induce the formation of E.coli O157:H7 VBNC in frozen food processing,and become one of the most potential potential safety hazards of quick-frozen food.Therefore,it is of great significance to determine the E.coli O157:H7 in the frozen beef meatball food system,which provides a scientific basis for the safety control of food borne pathogens.The main contents of this paper include three parts,respectively,the induction of VBNC E.coli O157:H7 in frozen beef pellets,the establishment of molecular biological detection method for VBNC E.coli O157:H7 and the transcriptome analysis of VBNC E.coli O157:H7.In this study,we established the low temperature induction system of frozen beef pellets at-20?and 4?respectively.After the induction at-20?for 152 days by plate counting method,E.coli O157:H7 in frozen beef pellets was confirmed to be non-culturable,The direct viable count method with Naphthyl keto acid was used for the verification that the non-culturable state of E.coli O157:H7 was viable,with a viable count of 6.3×10~7 cells/mL,but the number of culturable cells E.coli O157:H7 inducted at 4?was remained at 10~8 CFU/mL;We established a PCR molecular biological assay combining with propidium monoazide(PMA)and detected that the number of VBNC E.coli O157:H7 cells in frozen beef pellets was exceeded 10~7 cells/mL,achieving the transition from qualitative detection to semi-quantitative detection of routine PCR.Meanwhile,E.coli O157:H7 in frozen beef pellets was further verified to enter VBNC state by PMA-PCR method;The transcriptome of the low-temperature induction of VBNC E.coli O157:H7 cells and normal logarithmic growth phase cells were sequenced and analyzed by transcriptomics,and the significant differentially expressed genes were obtained.Through the GO function analysis,the differentially expressed genes were annotated and their functions predicted.Then the KEGG Pathway analysis was used to analyze and predict the metabolic pathways of the differentially expressed genes in the experiment,thus inferring the related molecular mechanism which was existented in VBNC E.coli O157:H7 under the conditions of low temperature induction.The research idea of this research is to start from the low temperature induction of VBNC state E.coli O157:H7,and to establish the rapid detection method of VBNC state cells.Finally,we use transcriptome analysis to explore the related molecular mechanisms in VBNC state cells.We hope that this research to provide effective and feasible reference for the detection of foodborne pathogens,thus providing scientific basis for solving the VBNC cell threat to human health and other security issues. |
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