Research On Rapid And Quantitative Detection Of Viable Common Foodborne Pathogens In Milk By PMA-qPCR

Food safety issues is one of the most concerned issues worldwide,many factors might result in food safety issues,among which foodborne pathogens is one of the major agents of food safety issues.Therefore,rapid and accurate quantitative detection of foodborne pathogens is of great significance for the prevention of food safety issues caused by foodborne pathogens.Fluorescence quantitative polymerase chain reaction(qPCR)is widely applied for rapid and quantitative detection of foodborne pathogens because of its speed and high throughput,nevertheless,which is not able of the quantitative detection of viable cells but also dead cells.Propidium monoazide(PMA)is a nucleic acid dye with high-affinity to DNA,which can eliminate the interference of dead cells on viable cell.In this study,rapid and quantitative detection methods were developed based on PMA-qPCR,and which were applied for the detection of common viable foodborne pathogens(Bacillus cereus,Staphylococcus aureus,Escherichia coli O157:H7,Salmonella spp.and Cronobacter spp.),aiming at providing technical support for rapid and accurate screening of foodborne pathogens.The content of each chapter of the paper is as shown as follows:The first chapter introduces the current situation of food safety issues caused by foodborne pathogens,and summarizes the relevant technologies of viable cell detection,and moreover reviews the research progress of PMA on viable cell detection.In chapter 2,a rapid and quantitative method based on PMA-qPCR has been established,and which was applied for rapid and quantitative detection of emetic B.cereus in milk.Results indicated that when viable cell concentration was in the range of 10~2-10~6 CFU/mL,which was linearly correlated with the Ct value of emetic B.cereus;Under optimal conditions,the LOD of emetic B.cereus was 10~2 CFU/mL in pure culture and artificially spiked milk.In chapter 3,a rapid and quantitative method based on PMA-qPCR assay was established,and which was applied for rapid and quantitative detection of viable E.coli O157:H7 and S.aureus in milk,respectively.The results showed that when the viable cells was in the range of 10~2 to 10~6 CFU/mL,which were closely correlated with the Ct value of E.coli O157:H7 and S.aureus;Under optimal conditions,the LOD of E.coli O157:H7 and S.aureus were 10~2 CFU/mL in pure culture and artificially spiked milk.In chapter 4,a rapid and quantitative method based on SD-PMA-mqPCR was established,and which was applied for simulate and quantitative detection of viable E.coli O157:H7,Cronobacter spp.and Salmonella spp.in milk.In this work,SD has been combined with PMA for eliminating the interference of the DNA from dead cells with intact membrane and dead cells with damaged membrane on viable cell detection.Results indicated that when viable cells were in the range of 10~2-10~7CFU/mL,which was linearly correlated with the Ct value of three cells.Under optimal conditions,the LOD of three cells were 10~2 CFU/mL in pure culture and artificially spiked milk.