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.
In this article, four types of standards were assessed in a SYBR Green-based real-time PCR procedure for the quantification of Staphylococcus aureus (S. aureus) in DNA samples. The standards were purified S. aureus genomic DNA (type A), circular plasmid DNA containing a thermonuclease (nuc) gene fragment (type B), DNA extracted from defined populations of S. aureus cells generated by Fluorescence Activated Cell Sorting (FACS) technology with (type C) or without purification of DNA by boiling (type D). The optimal efficiency of 2.016 was obtained on Roche LightCycler(®) 4.1. software for type C standards, whereas the lowest efficiency (1.682) corresponded to type D standards. Type C standards appeared to be more suitable for quantitative real-time PCR because of the use of defined populations for construction of standard curves. Overall, Fieller Confidence Interval algorithm may be improved for replicates having a low standard deviation in Cycle Threshold values such as found for type B and C standards. Stabilities of diluted PCR standards stored at -20°C were compared after 0, 7, 14 and 30 days and were lower for type A or C standards compared with type B standards. However, FACS generated standards may be useful for bacterial quantification in real-time PCR assays once optimal storage and temperature conditions are defined.
Traditional detection methods for Enterobacteriaceae in foods are time-consuming and laborious. The current study assessed the specificity of three real-time PCR primer sets. Set A (IEC primers) targeted the conserved flanking regions of the 16S rRNA, the 16S-ITS-23S gene region. Set B (ENT primers) annealed to Escherichia coli 16S ribosomal RNA gene. The third set (C) used a D-LUX™ (Light Upon eXtension) single FAMlabelled forward primer and a corresponding unlabeled primer. Set A was specific for E. coli and for some nonEnterobacteriaceae. SYBR Green-based real-time PCR confirmed the specificity of set B for the Enterobacteriaceae but also detected Vibrionaceae. In contrast, set C was poorly specific. However, set D including the forward LUX™ primer from set C and the reverse primer from set B had a specificity comparable to that of set B, but with higher sensitivity. This combined set was successfully applied to detect Enterobacteriaceae in infant milk formula and compared favourably with a commercial real-time PCR kit.
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