Establishment And Application Of Multiplex Tandem PCR For Detection Of 7 Pathogenic Bacteria

Dairy products are loved by people all over the world for their rich nutrients,and they are also a paradise for microbial reproduction,making dairy products easy to be contaminated by bacteria and causing many food safety problems.Detection of common food-borne pathogens in dairy products is great significance for controlling the outbreak of food-borne diseases and safeguarding food safety in China.Traditional microbial detection methods mainly rely on medium culture,biochemical serum identification,morphological characteristics observation,etc.,these methods are cumbersome steps,time-killer,unable to quantitative and unable to detect a variety of pathogenic bacteria simultaneously.Therefore,it is necessary to establish a rapid,effective,high-throughput,quantitative detection method to meet the current development trend.The purpose of this study is to establish Multiplex-tandem quantitative PCR assay that can simultaneously detect 7 kinds of food-borne pathogenic bacteria and can be applied to different actual samples.At the same time,the feasibility of this method is evaluated by actual factory sampling and simulating the samples through manual poisoning.The main research contents and results are as follows:1.Establishment of Multiplex-tandem PCR assaySelect OmpA gene of Sakazakii and ORF2819 genes of listeria,the uidA gene of E.coli,the Nuc gene of Staphylococcus aureus,STM4497 gene of Salmonella typhimurium,nheA gene of Bacillus,RFC gene of Shigella set up Multiplex-tandem quantitative PCR assay and detected 7kinds of foodborne pathogenic bacteria;The specificity test and standard curve showed that this method performed perfect specificity and amplification efficiency.At the same time,the key point of this assay:the number of cycles in the first round was optimized by compared with normal real-time quantitative PCR,it was found that when the number of cycles in the first round was higher than 15,the amplification effect was greater than normal real-time quantitative PCR.However,considering that the number of cycles increased,the number of non-specific amplification products also increased,the optimal number of cycles was set as 15.At the optimal cycle number,this method can simultaneously detect the DNA of staphylococcus aureus of 10^-3ng/μL,10^-4 ng/μL of Listeria,and the lower limit of detection for the remaining six strains reaches 10^-5 ng/μL.2.Evaluation of the detection ability of Multiplex-tandem quantitative PCRIn order to evaluate the sample detection ability of MT-PCR,10-fold series diluents of 7target strains were artificially added into sterile water,sterile infant formula powder and instantaneous Ultra Heat-Treated sterilized milk as samples to be tested.The results showed that the method could detect 7 target strains of 10¹ CFU/mL in sterile water.10² CFU/mL of 7 target strains can be detected in Ultra Heat-Treated milk.In infant formula powder,10² CFU/g could be detected in listeria monocytogenes and staphylococcus aureus,and 10³ CFU/g could be detected in all other strains.3.Application of Multiplex-tandem quantitative PCR in actual factory detectionIn order to apply this method to the actual detection,this study sampling from an infant rice noodle factory in Guangzhou,analyzed the factory samples with this method,and compared with Sond generation sequencing and traditional microbial detection analysis.The results showed that there were some pollution points that could be easily ignored in factory,such as the residual powder in the WPS air duct in the drum.The sampling point had high microbial abundance,was difficult to disassemble and clean,and accumulated residual powder for a long time,which was the area prone to outbreak of microbial pollution.By comparing the three detection methods,the traditional microbial detection method can only preliminarily determine that the sampling point is a high-risk area,and it is unable to determine the quantity and type of contaminated bacteria quantitatively,which also takes a long time.The Sond-generation high-throughput detection technology has a high flux and can detect the types and abundance of microorganisms in the residual powder and water samples,but it is unable to quantify the microorganisms specifically.Moreover,multiple tandem real-time quantitative PCR could accurately identify the microorganisms that were involved in the key pollution,with quantified these microorganisms.It was finally found that staphylococcus aureus only existed in dust sample no.37 and P sample,with a colony number of 278 CFU/g and 266 CFU/g,respectively.Escherichia coli was found in samples 4-2,4,6-2,6,P and 37,with number of 1.8×10⁵ CFU/g,2.9×10⁵ CFU/mL,3.7×10⁴CFU/g,4.6×10⁵ CFU/mL,5.2×10⁵ CFU/g and 4.4×10⁴ CFU/g,respectively.Except samples no.4 and no.37,shigella was found in samples 4-2,6-2,6,and P,with number of 432 CFU/g,912CFU/g,623 CFU/mL,and 884 CFU/g,respectively.