Estabilishment And Applicaion Of Multiplex PCR For Detecion Of Four Foodborne Bacterial Pathogens

There is a growing concern for foodborne pathogens about human disease and death. Citizens have more and more demands to ensure their food suppliment are safe and edible. These demands make food testing methods must continue to progress.The traditional methods are slow, low accuracy and needed for highly professional trained person to complete. The emergence of biotechnology has greatly changed the food testing methods. A positive impact on the field of immunology, molecular biology, automation and information technology has been seen. We should actively explore the possibility of combinition about different rapid methods. The latest detection technology is moving to quantitative detection of target organisms and the simultaneous detection of multiple pathogen or toxin.Compared with conventional PCR, Mulptiple PCR has a higher level of priciple. More than two pairs of primers were added in the same reaction system. Each primer pairs could amplify the respective product at the same time. By agarose gel electrophoresis, we can get the rusults at the same time as well.To establish a method of diagnosing four species food-borne pathogens synchronous by multiplex PCR. Previously, the molecular biology detection method does not exist for Citrobacter freundii. Four pairs of suitable Primers were designed according to the specific gene fimY of Salmonella spp., the gene ldh of Citrobacter freundii, the gene idsC of Proteus mirabilis, the gene fimA of Edwardsiella tarda. The amplified fragment length of PCR was161bp,336bp,396bp,526bp. Firstly, we set up four single PCR detection methods for confirm their specificity and sensitivity. The results displayed their identity up to99%, and sensitivity of Salmonella spp. up to89CFU; of Citrobacter freundii up to72CFU; of Proteus mirabilis up to6CFU; of Edwardsiella tarda up to7CFU. Thus the single PCR detection method provided a good reference for the multiplex PCR detection method. In the process of establishing the multiplex PCR detection method, each major influnce factor of reaction has been optimized separately, such as annealing temprature, primer consistence, dNTP and Mg2+concentration. The results indicated that all of four strains of bacteria have specific brightly stripes. Sensitivity of Salmonella spp.up to89CFU; of Citrobacter freundii up to7CFU; of Proteus mirabilis up to57CFU; of Edwardsiella tarda up to6CFU.The detection for man-made pollution samples proved that the multiplex PCR method has the advantages of reliable, sensitive, and rapid. The detection limit was beneath than10CFU/ml after pre-enrichment step.To sum up, the detection method is higly suitable for the detection of four foodborne enteric pathogens in he food processing and food import and exprot sectors. It has a wide application space. In addition, it provide the key technical support for developing a kit that detects the four species food-borne pathogens rapidly.At the same time,the study also explored the feasibility of matrix-assisted laser desorption ionization time of flight mass spectrometer detection method of the four bacterias, compared with established single PCR method, we can fi nd that this method has high specificity, easy operation advantages.