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 Staphyloccocus Aureus in Foods

Abstract

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. PRACTICAL APPLICATIONS 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.

Development of Defined Microbial Population Standards Using Fluorescence Activated Cell Sorting for the Absolute Quantification of S. aureus Using Real-Time PCR

“…SYBR Green I was included as detection format for its simplicity, low cost, and as binding is not affected by mutation of the target gene [5], to avoid increased handling and possible loss of detection found for probes such as TaqMan Ò or HybProbes [34,35]. Indeed, data obtained in the study of Martinon and Wilkinson [26] confirmed that the primer set to target the nuc gene was highly specific for S. aureus and that SYBR Green I was a suitable detection format. Times and temperatures applied are displayed on Table 1, and DNA amplification was followed in real-time in channel F1 or at 530 nm.…”

“…Therefore, the development of a rapid and quantitative real-time PCR detection system for this pathogen is of both industrial and public health interest. A number of studies have described realtime PCR assays for quantification of S. aureus in various foods using genomic or plasmid DNA standards [22][23][24][25][26]. Moreover, real-time PCR systems for the quantitative detection of S. aureus can be applied to clinical diagnosis, as described by Peters et al [27], for the determination of bacteremia.…”

Swab sample preparation and viable real-time PCR methodologies for the recovery of Escherichia coli, Staphylococcus aureus or Listeria monocytogenes from artificially contaminated food processing surfaces

“…To date, we have been working on a number of optimized realtime PCR protocols to enable the detection of Enterobacteriaecae (Martinon, Cronin, & Wilkinson, 2011a), S. aureus or L. monocytogenes in a range of foods (Martinon & Wilkinson, 2011). The objectives of this study were to develop sample preparation and viable real-time PCR procedures for the quantitative recovery of foodborne pathogens such as E. coli, S. aureus or L. monocytogenes in swabs collected from contaminated food contact surfaces.…”

Aptasensors for Staphylococcus aureus Risk Assessment in Food

“…After adding the total DNA of the object to be tested, the sequence of the target gene of SEB is amplified by PCR or real-time fluorescence PCR, and quantitative monitoring can be conducted by electrophoresis bands or fluorescence intensity. Martinon and Wilkinson (2011) established a low-cost, SYBR Green based double-chain real-time PCR for simultaneous detection of Listeria monocytogenes and S. aureus in food. Detection limit was 2 CFU/g in raw meat containing S. aureus ( Martinon and Wilkinson, 2011 ).…”

“… Martinon and Wilkinson (2011) established a low-cost, SYBR Green based double-chain real-time PCR for simultaneous detection of Listeria monocytogenes and S. aureus in food. Detection limit was 2 CFU/g in raw meat containing S. aureus ( Martinon and Wilkinson, 2011 ). However, PCR-based tests used alone are unable to provide any indication of the cell viability being examined because they do not distinguish DNA from living and dead cells ( Foddai and Grant, 2020 ).…”