Establishment Of Multiple Quantitative Pcr For Detection Of Food-borne Pathogens

Food safety is regarded as a global public health problem.Foodborne pathogens are important aspects of food safety.In this paper,five kinds of food-borne pathogens were studied by Salmonella,Enterobacter sakazaki,Vibrio parahaemolyticus,Listeria monocytogenes and Staphylococcus aureus.Five qPCR systems and a triplex qPCR system were established,and a fluorescence quantitative PCR automatic testing system was established,which was developed independently by microbiological agents of microbiological agents in Huzhou and the Key Laboratory of agricultural product safety.The reliability of the above test methods is confirmed by the experiments of sample simulation,real food inspection and traditional methods.1.The primers and probe sequences of five foodborne pathogenic bacteria in this paper were derived from SN/T 1870-2007.After optimizing the probe concentration in the single detected system,a single qPCR detection system for five kinds of pathogenic bacteria of Salmonella,Staphylococcus aureus,Listeria monocytogenes,Vibrio parahaemolyticus and Enterobacter sakazaki was established.The limit of minimum detection was 69 copies/m L in artificial simulated milk,limit of minimum detection was 37 copies /m L in artificial simulated bread,and the reproducibility of the five single qPCR systems was good,and coefficient of variation in the group was 1.30%~3.89%.2.We established a triplex qPCR detection system for simultaneous detection of Salmonella,Listeria monocytogenes and Vibrio parahaemolyticus.Through the bread sample simulation experiment,the limit of minimum detection of Salmonella,Listeria monocytogenes and Vibrio parahaemolyticus in the triplex PCR detection system reached 7 copies/m L,32 copies/m L and 42 copies/m L,respectively.The repeatability of the three test system and the coefficient of variation among the groups were also analyzed.The number is between0.52% and 2.18%,which indicating that the detection system has good repeatability.3.The 14 samples of food samples were detected by single qPCR,and 40 seafood samples were detected by triplex qPCR detection.The results showed that the system could detect food borne pathogens accurately.For single detection,only Db2 detected Salmonella in quantitative detection.In qualitative tests,Salmonella was suspected in Db2,Da3 and Xa2 samples.The detection rate of three tests was 71.67%,the detection rate of national standard method was 82.5%,the correct rate of Salmonella in the three qPCR detection methods was 100%,the correct rate of Vibrio parahaemolyticus was 50%,and the correct rate of single increase of Listeria monocytogenes was 32%.In this study,a triplex qPCR detection method and a testing system matching with microbiological detector are set up,and a fully closed and integrated automatic testing system can be developed to meet the demand of single weight detection.It provides a solid foundation for rapid response of foodborne pathogens in food.