Development Of Multiplex Real-time PCR For Pathogen Detection And Its Application In Milk

Staphylococcus aureus(S.aureus),Listeria monocytogenes(L.monocytogenes)and Salmonella spp.are the major foodborne pathogens,and the food poisoning accidents caused by these three pathogens occurrs frequently,which are harmful to human and public health.Milk is a widely loved food in the world,and because of its abundant nutrients,milk is easily contaminated with bacteria.Detection of the major foodborne pathogens in milk is of great importance in controlling foodborne outbreaks and ensuring food safety.Therefore,building a set of efficient,rapid and specific detection methods is in line with the current development trend.This study aims to establish a set of real-time PCR detection methods that can be applied to the detection of major pathogens in milk in different practical situations and to explore the inhibitory effect of lactic acid bacteria on the growth of pathogenic bacteria,which is in order to provide some technical guidance on detection of major pathogens in milk.The main research contents and results are as follows:1.Development of a multiplex real-time PCR method with pre-enrichment for detection of major pathogens in milkThe nuc gene of S.aureus,the hlyA gene of L.monocytogenes and the orgC gene of Salmonella were selected as the targeted gene to bulid the multiplex real-time PCR for simultaneous detection of S.aureus,L.monocytogenes and Salmonella in milk.The standard curve shows that the method has good specificity and amplification efficiency.On the basis of multiplex real-time PCR,Biocreen and the predictive model were used to optimize the enrichment process.Brain Heart Infusion(BHI)broth was determined to be the best broad-spectrum bacteriostatic solution,and the recommended enrichment time was 4 h.The minimum detection limit of this method after optimization by the pre-enrichment step was 12 CFU/25 mL for S.aureus,14 CFU/25 mL for L.monocytogenes and 10 CFU/25 mL for Salmonella.The multiplex real-time PCR method with pre-enrichment was successfully applied to the detection of 46 samples of raw milk and its pasture environment,which proved the value of this method in practical application.The optimized real-time PCR method has the advantages of low detection limit,high specificity and shortened detection time,which meets the needs of actual sample detection.2.Development of PMA-based multiplex real-time PCR method for pathogen detection in milkOn the basis of multiplex real-time PCR method,the application of PMA in the detection of viable cells was studied.For PMA treatment conditions,25 uM was determined to be the optimum PMA concentration by the inhibitory effect of PMA on the DNA of dead cells,meanwhile,the signal of 103 CFU/mL viable cells could be detected in the presence of DNA of 107 CFU/mL dead cells.The optimized PMA treatment combined with multiplex real-time PCR was established to simultaneously detect S.aureus,L.monocytogenes and Salmonella in milk.Specific experiments and standard curves show that the detection method based on PMA has good specificity and amplification efficiency,and the minimum detection limit of this method in milk is 102-103 CFU/mL.The real-time PCR detection method based on PMA is a substitute and compensation of traditional culture methods and ordinary PCR methods.3.Application of multiplex quantitative real-time PCR in studying competition growth among strainsThe nuc gene of S.aureus and the 16s rDNA gene fragment of Lactobacillus acidophilus were selected to construct two real-time fluorescence quantitative PCR methods for simultaneous detection of lactic acid bacteria(LAB)and S.aureus in milk.The constructed standard curve shows that the amplification efficiencies of lactic acid bacteria and S.aureus in milk are 92.3%and 105.4%,respectively.At the same time,the method was successfully applied to explore the growth inhibition effect of LAB on S.aureus in milk at different temperatures.By analyzing the growth curves and growth parameters fitted by Baranyi model,we find out that LAB could inhibit the growth of S.aureus at 37 ℃,and metabolites of LAB reduced the pH in the milk and caused the DNA of S.aureus dissolved.LAB inhibited the growth of S.aureus at 25 ℃,but its inhibition effect was weak.At 4 ℃ LAB and S.aureus grew slowly,and LAB had no inhibitory effect on the growth of S.aureus.The results provided some theoretical support for the actual production and prevention of S.aureus.