Detection Technology Research Of Common Food-borne Bacterial Pathogens In Chilled Meat By Multiplex PCR Assay

Salmonella,Escherichia coli O157:H7 and Listeria monocytogenes are three food-borne bacterial pathogens,they might contaminate meat and cause deadly diseases in humans.The conventional methods for the ditection of them require multiple subculture steps followed by biochemical and serological confirmation,which may take up to 5-7 days.The immunological method was proved to be simple and rapid when compared with standard culture method,but it might cause false-positive or false-negative result.3 pairs of primers were designed by primer 5 and Oligo software,in which the segments of the hns gene,eaeA gene and hly gene were specifically amplified and a novel method of multiplex PCR assay was developed.The selected primers amplified fragment size of 534bp,422bp,and 273bp for Salmonella,Escherichia coli 0157:H7,and Listeria monocytogenes,respectively.Non-specific PCR products were not detected.This assay provides specific and reliable results and allows for the cost-effective detection of all three bacterial pathogens in one reaction tube.The primers chosen for the multiplex PCR had great relationship with virulence factors. Have knowledge with these virulence factors,we could set up new methods and new technology further which would be helpful to rapid,accurate,molecular identification of pathogenic bacteria in meat.There are two methods for DNA extraction.One is Paeonol/chloroform method, the other is boiling.The amplified fragment of DNA by Paeonol/chloroform method was brighter than the boiling method.It is suggested that Paeonol/chloroform is an available method aimed for pathogens' DNA extraction.Through optimizing the system and condition of muhioplex-PCR,it could concurrently detect the three pathogens at 2.3×10~2 cfu/mL within 6h,got the single, steady and clear result when the method was applied primarily in the analysis of artificially inoculated meat samples.The sensitivity below the single PCR's,but above the affection dose of the three pathogens.It was significant in ensuring the ment security.The established assay had been applied to plenty of meat or artificially inoculated meat samples.Excellent agreement was obtained for the results of multiplex PCR and the conventional culture method,which suggests that the multiplex PCR is a reliable and useful method for rapid screen for the presence of Salmonella,Escherichia coli 0157:H7,and Listeria monocytogenes in meat.