Simultaneous Detection Of Salmonella, Listeria Monocytogenes,and Escherichia Coli O157:H7in Beef By Multiplex PCR

Foodborne pathogens remain a major public health concern. Foodborne illness caused by bacteria account for the largestnumber and involved most people.Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7are considered major foodborne pathogens.The beef have been associated with out-breaks caused by Salmonella, Listeria monocytogenes, and Escherichia coli O157:H7. Reliable detection techniques are a prerequisite for identification of these pathogens in foods, food sources, and food processing plants. Conventional culture methods for detection of pathogens needed for the different operations (enrichment, isolation of colonies and identification), they often result in time consuming, analyses not always compatible with the need for rapid results. Nucleic acid based amplification techniques, of which the multiplex PCR method has been so far the most extensively employed, high-effective, reliable and useful tool in testing three food-borne bacterial pathogens.A lot of reported primer combinations were tested in pre-experient in order to select the high specificity and low cross-contamination primers. SalmonellainvA gene, a virulence gene encoding an invasion protein and exclusively exists in almost all Salmonella spp. hlyA was selected to discriminate the Listeria monocytogenes from the Listeria spp.O-antigen biosynthesis loci (rfbE) is conserved in E. coliO157with non-H7flagellar antigens, in combination with the flagellar antigens, to direct detection of E. coli O157:H7. The results showed that all Salmonalla isolates yielded a284bp; Listeria monocytogenes yielded a456bp; O157yielded612bp,825bp. Each primer generated the expected size, indicating PCR was used as reliable means of detection. Each pathogen and a combination of few individual strains were used to test PCR specificity. Results of the multiplex PCR assay were consistent with our expectation.The optimized reaction conditions followed as the concentration of primer0.2μmol/L for Salmonella spp.,0.1μmol/L for Listeria monocytogenes,1μmol/L(flic) and0.3μmol/L(rfbE) for E.coli O157:H7,4mmol/L Mg2+,0.4mmol/L dNTP,0.5UTaq DNA polymerase,1μLDNA templet. PCR conditions in a thermal cycler were as follows:an initial denaturation at95℃for30s followed by35cycles of amplification at95℃for30s, annealing at58℃for30s; extension at72℃for30s; and a final extension at72℃for7min. Under the condition, the detection limits was102CFU/mL for Salmonella and Listeria monocytogenes,103CFU/mL for E.coli O157:H7in pure cultures. After18h enrichment in artificially contaminated beef samples, the detection limits of Salmonella and E.coli O157:H7was100CFU/g, Listeria monocytogenes was102CFU/g in the multiplex PCR assay.Various extraction methods have shown different extraction efficiency in the performance of PCR. Comparing with the boiling method and Phenol-chloroform method, the BacteriaGen DNA Kit had higher yields by the PCR assay, demonstrating that the protocol to be an effective way to yield satisfactory bacterial DNA and eliminate inhibitors from artificially contaminated ground beef. Comparing the enrichment effeciency of TSB and LB medium, the LB was used as an enrichment medium in the multiplex PCR. The results demonstrated that after18h enrichment in LB, the multiplex PCR assay was able to simultaneous identification of these3pathogens down to102CFU/g in ground beef, the detection limits of Salmonella and E.coli O157:H7was100CFU/g, Listeria monocytogenes was102CFU/g.In order to assess the actual use of multiplex PCR assay to detect the three pathogens in beef, this method and traditional method were compared. It is indicated that the multiplex PCR have higher sensitivity,specificity and coincidence rate for detection of the actual samples of the three pathogens. The multiplex PCR method we developed for detecting Salmonella, Listeria monocytogenes and E. coli O157:H7in ground beef has a strong practical value in controlling pathogen transmission.