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Selection of optimal primer sets for use in a duplex SYBR green-based, real-time polymerase chain reaction protocol for the detection of Listeria monocytogenes and Staphylococcus aureus in foods

Martinon, A., Wilkinson, M.G.
Journal of food safety 2011 v.31 no.3 pp. 297-312
Listeria monocytogenes, Staphylococcus aureus, annealing, dairy products, detection limit, enriched foods, food analysis, food industry, gene targeting, genes, good hygiene practices, hazard characterization, melting point, microbiological criteria, microorganisms, plate count, polymerase chain reaction, raw meat, ready-to-eat foods, screening
A low-cost duplex SYBR Green-based, real-time polymerase chain reaction (PCR) for the simultaneous detection of Listeria monocytogenes and Staphyloccocus aureus in foods was developed following selection of optimal primers. For L. monocytogenes, the set targeting the listeriolysin O gene (hlyA primers) was more specific than the one annealing to the metalloprotease gene (mpl primers). For S. aureus, the nuc primers targeting the thermonuclease gene were highly specific. Simplex SYBR Green-based, real-time PCR methods for the separate detection of L. monocytogenes and S. aureus were performed. Finally, the developed duplex real-time PCR was applied to foods spiked with these microorganisms using a simple enrichment step in buffered peptone water at 37C for 18 h. Melting temperatures were sufficiently different for identification with intra and inter-assay coefficients of variation in melting temperature of 0.08% and 0.20%, respectively. Detection limits were 7 colony-forming unit (cfu)/g in coleslaw for L. monocytogenes and 2 cfu/g in raw minced meat for S. aureus, as confirmed using the commercial kits and plate counting. This multiplex real-time polymerase chain reaction method provides a potential two-in-one screening enabling simultaneous detection of positive samples of S. aureus and L. monocytogenes in minimally enriched food samples. Considering the simplicity, low cost, rapidity and acceptable reproducibility shown for the detection of S. aureus and L. monocytogenes, this duplex method may be used in food analysis prior to further potentially more expensive investigations by giving an initial indication of contamination and discarding of negative samples. This method may contribute to the validation and the verification of hazard analysis critical control plans, good hygiene practices and the acceptability of batches in food industries. In regard to European Community microbiological criteria regulation, this alternative method may be applied especially for the analysis of L. monocytogenes in ready-to-eat foods and S. aureus in dairy products.