NOVEL MULTIPLEX PCR ASSAY FOR RAPID DETECTION OF FIVE BACTERIAL FOODBORNE PATHOGENS
Keywords:
Foodborne pathogens, milk, multiplex PCR, target genesAbstract
Milk and dairy products can harbor varieties of foodborne pathogens especially Bacilluscereus, Escherichia coli, Listeria monocytogenes, Salmonella spp., and Staphylococcusaureus. In this work, a rapid multiplex polymerase chain reaction (m-PCR) method forsimultaneous detection of 5 major foodborne pathogens in milk was developed. Specificprimers targetting the enterotoxin FM, uspA, prfA, fimY, and eap genes were selected forspecific detection of B. cereus, E. coli, L. monocytogenes, Salmonella spp., and S. aureus,respectively. The optimum concentrations of the primers in the m-PCR reaction were 0.04μM enterotoxin FM, 0.12 μM uspA, 0.16 μM prfA, 0.04 μM fimY, and 0.2 μM eap. Theexpected polymerase chain reaction (PCR) products of 513, 884, 398, 315, and 230 bp were detected from the specific amplification of B. cereus, E. coli, L. monocytogenes, Salmonellaspp., and S. aureus, respectively. Cross-amplifications from non-target bacteria isolatedfrom raw milk samples were not detected. The developed m-PCR methods could detect all5 target bacteria at the level of at least 100 ng of each from mixed genomic DNA extracted from pure cultures. These results indicated that the developed m-PCR using 5 primer setscan be used for B. cereus, E. coli, L. monocytogenes, Salmonella spp., and S. aureusdetection with no cross-reactivity with other non-target bacteria found in the enrichmentculture. For future work, the m-PCR technique will be applied to detect multiple foodborne pathogens in enrichment cultures from milk samples with considerable timesaving andcost-effectiveness compared with the biochemical characterization of the conventional method.
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