title: A multiplex real-time polymerase chain reaction for simultaneous detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products.
abstract: To achieve an effective detection of Salmonella spp., Escherichia coli O157, and Listeria monocytogenes in meat products, a multiplex real-time polymerase chain reaction (PCR) coupled with a multipathogen enrichment strategy was developed in this study. Pathogen-specific DNA sequences in the invA, rfbE, and hlyA genes were employed to design primers and TaqMan probes for identifying Salmonella spp., E. coli O157, and L. monocytogenes, respectively. An internal amplification control (IAC) utilizing a novel DNA sequence from human adenovirus was incorporated into the multiplex PCR assay to indicate false-negative results. Concurrent amplifications of multiple targets and IAC were thoroughly evaluated and optimized to minimize PCR competitions. Combined with a multipathogen enrichment in a selective enrichment broth for Salmonella, Escherichia, and Listeria (SEL), the multiplex real-time PCR assay was able to simultaneously detect all of the three organisms in artificially contaminated ground beef at a detection sensitivity of <18 CFU/10 g ground beef. Applying the assay to 26 retail meat samples including beef, chicken, turkey, and pork revealed that 12 samples were positive for one of the organisms and 3 samples were positive for two of the organisms after a 20-h enrichment in SEL. The remaining meat samples tested negative for all of the organisms by only showing amplification of the IAC. These results were confirmed by traditional culture methods testing for each individual species. Taken together, the multiplex real-time PCR assay combined with multipathogen enrichment is a rapid and reliable method for effectively screening single or multiple pathogen occurrences in various meat products.
authors.affiliation: Joint Sino-U.S. Food Safety Research Center and Bor Luh Food Safety Center, School of Agriculture and Biology, Shanghai Jiao Tong University, Shanghai, China.
authors.forename: Biao
authors.initials: B
authors.lastname: Suo
authors.forename: Yiping
authors.initials: Y
authors.lastname: He
authors.forename: Shu-I
authors.initials: SI
authors.lastname: Tu
authors.forename: Xianming
authors.initials: X
authors.lastname: Shi
mesh_terms: D000818:Animals; D001426:Bacterial Proteins / Q000235:genetics / Q000378:metabolism; D015373:Bacterial Typing Techniques; D001431:Bacteriological Techniques; D002238:Carbohydrate Epimerases / Q000235:genetics / Q000378:metabolism; D002417:Cattle; D004269:DNA, Bacterial / Q000302:isolation & purification / Q000378:metabolism; D019453:Escherichia coli O157 / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D005517:Foodborne Diseases / Q000517:prevention & control; D005798:Genes, Bacterial; D006460:Hemolysin Proteins / Q000235:genetics / Q000378:metabolism; D057230:Limit of Detection; D008089:Listeria monocytogenes / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D008461:Meat Products / Q000382:microbiology*; D008462:Meat-Packing Industry / Q000379:methods; D016133:Polymerase Chain Reaction / Q000379:methods*; D011200:Poultry / Q000382:microbiology; D012475:Salmonella / Q000145:classification / Q000235:genetics / Q000254:growth & development / Q000302:isolation & purification*; D013045:Species Specificity; D034421:Sus scrofa / Q000382:microbiology; D000637:Transaminases / Q000235:genetics / Q000378:metabolism
chemical_list: D001426:Bacterial Proteins; D004269:DNA, Bacterial; D006460:Hemolysin Proteins; C075518:invA protein, Bacteria; D000637:Transaminases; D002238:Carbohydrate Epimerases; C061272:perosamine synthetase