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Detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus using duplex real-time PCR assay with melting curve analysis on fresh lettuce  

Lee, Na-Ri (Food Safety Research Division, Korea Food Research Institute)
Kwon, Kyung-Yoon (Food Safety Research Division, Korea Food Research Institute)
Choi, Sung-Wook (Food Safety Research Division, Korea Food Research Institute)
Koo, Min-Seon (Food Safety Research Division, Korea Food Research Institute)
Chun, Hyang-Sook (Food Safety Research Division, Korea Food Research Institute)
Publication Information
Journal of Food Hygiene and Safety / v.26, no.2, 2011 , pp. 114-119 More about this Journal
Abstract
In this study, two duplex real-time PCR approach with melting curve analysis is presented for the detection of Escherichia coli O157:H7, Listeria monocytogenes, Salmonella spp. and Staphylococcus aureus, which are important food-borne bacterial pathogens usually present in fresh and/or minimally processed vegetables. Reaction conditions were adjusted for the simultaneous amplification and detection of specific fragments in the ${\beta}$-glucuronidase (uidA, E. coli), thermonuclease (nuc, S. aureus), hemolycin (hly, L. monocytogenes) and tetrathionate reductase (ttr, Salmonella spp.) genes. Melting curve analysis using a SYBR Green I real-time PCR approach showed characteristic $T_m$ values demonstrating the specific and efficient amplification of the four pathogens; $80.6{\pm}0.9^{\circ}C$, $86.9{\pm}0.5^{\circ}C$, $80.4{\pm}0.6^{\circ}C$ and $88.1{\pm}0.11^{\circ}C$ for S. aureus, E. coli O157:H7, L. monocytogenes and Salmonella spp., respectively. For all the pathogens, the two duplex, real-time PCR was equally sensitive to uniplex real-time PCR, using same amounts of purified DNA, and allowed detection of 10 genome equivalents. When our established duplex real-time PCR assay was applied to artificially inoculated fresh lettuce, the detection limit was $10^3$ CFU/g for each of these pathogens without enrichment. The results from this study showed that the developed duplex real-time PCR with melting curve analysis is promising as a rapid and cost-effective test method for improving food safety.
Keywords
Real-time PCR; Multiplex; Melting curve; Fresh; minimally produced vegetable; Food borne pathogens;
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