Research Of Rapid Detection Method For Viable Cells Of Three Foodborne Pathogens In Dairy Products And Application Of The Detection Kits

Foodborne pathogens such as Salmonella(sal),Bacillus cereus,and Cronobacter sakazakii in milk and dairy products are important causes of mass food poisoning,which could cause irreparable damage to lives and property of people.The traditional national standard test method is the bacteriology culture method,which include enrichment culture,selective separation,morphological characteristic observation,biochemical reaction and serological identification,the procedure is tedious and consumes.Therefore,it is of great significance to establish a fast and effective detection method to control the contamination of foodborne pathogens in milk and dairy products.Multiple polymerase chain reaction(mPCR)has been widely used in the detection of foodborne pathogens in food substrates because of its simple operation,strong specificity,high sensitivity and short time consuming.Propidium monoazide(PMA)can eliminate the interference of dead bacteria,and detect only viable bacteria,which can effectively eliminate the possible"false positive"results.In this study,a PMA-mPCR method for rapid detection of three food borne pathogenic bacteria in milk and dairy products was established.The DNA was extracted from Sal,B.cereus and C.sakazakii,respectively,with Sal invA,cesB of B.cereus,and OmpA of C.sakazakii.The multiplex PCR method for detecting the 3strains of viable bacteria can be detected.Carried out with mPCR technology,the high concentration and sensitive detection of Sal,B.cereus and C.sakazakii in milk and dairy products was achieved.The contents of each chapter are described as follows:The first chapter summarizes the research progress of the detection technology of common foodborne pathogens in dairy products.The second chapter established the PMA-mPCR method for detecting Sal,B.cereus and C.sakazakii in dairy products,the experimental results show that for C.sakazakii OmpA,Salmonella Enteritidis invA and B.cereus cesB primers have better specificity;and PMA with concentration of 10g/mL was effective in inhibiting the PCR amplification of the DNA of 10~7CFU/mL S.Enteritidis,B.cereus and C.sakazakii,and no effect on the living bacteria DNA.The detection limit in pure culture of S.Enteritidis,B.cereus and C.sakazakii was 10~4CFU/m L;in standard samples of dairy products,for after 7 h culture,the detection limit of S.Enteritidis,B.cereus and C.sakazakii was 10~0 CFU/mL.The PMA-mPCR method established in this study has realized the rapid detection method of C.sakazaki,Sal and B.cereus in dairy products.The third chapter compared the traditional method of national standard method and PMA-m PCR.The PMA-mPCR method can be well applied in food inspection.And can significantly shorten the testing time,multiple detection of foodborne pathogenic bacteria for dairy products provide a fast accurate efficient economic means of detection.A total 1165 batches of dairy products samples were tested using the established method,and found that the infant milk powder still exist for C.sakazakii in the risk of pollution.The method established in this work has also provided a good testing tool for infant milk.In the fourth chapter,the multiplex PCR detection kit for C.sakazaki,sal and B.cereus in dairy products was developed.According to the actual testing requirement,the reagent used in the multiplex PCR detection method is assembled into a suitable kit,and the instructions for the complete process of the sample test kit are attached.The kit can be kept for 12 months at-20℃,but should avoid repeated freezing and thawing.The kit has better repeatability and stability,while maintaining a high sensitivity and high specificity of conventional multiple PCR,and simplifies the operation,keep the high stability and repeatability,suitable for dairy products for C.sakazaki,sal and B.cereus batch testing,and thus has broad market prospect and application value.