Application of 5′-Nuclease PCR for Quantitative Detection of Listeria monocytogenes in Pure Cultures, Water, Skim Milk, and Unpasteurized Whole Milk

Abstract

PCR techniques have significantly improved the detection and identification of bacterial pathogens. Countless adaptations and applications have been described, including quantitative PCR and the latest innovation, real-time PCR. In real-time PCR, e.g., the 5-nuclease chemistry renders the automated and direct detection and quantification of PCR products possible (P. M. Holland et al., Proc. Natl. Acad. Sci. USA 88:7276-7280, 1991). We present an assay for the quantitative detection of Listeria monocytogenes based on the 5-nuclease PCR using a 113-bp amplicon from the listeriolysin O gene (hlyA) as the target. The assay was positive for all isolates of L. monocytogenes tested (65 isolates including the type strain) and negative for all other Listeria strains (16 isolates from five species tested) and several other bacteria (18 species tested). The application of 5-nuclease PCR in diagnostics requires a quantitative sample preparation step. Several magnetic bead-based strategies were evaluated, since these systems are simple and relatively easy to automate. The combination of nonspecific binding of bacteria to paramagnetic beads, with subsequent DNA purification by use of the same beads, gave the most satisfactory result. The detection limit was approximately 6 to 60 CFU, quantification was linear over at least 7 log units, and the method could be completed within 3 h. In conclusion, a complete quantitative method for L. monocytogenes in water and in skimmed and raw milk was developed.

Rapid real-time PCR detection of Listeria monocytogenes in enriched food samples based on the ssrA gene, a novel diagnostic target

“…Recently conventional and real-time PCR assays have been developed for the detection of L. monocytogenes in foods (Nogva et al, 2000;Hein et al, 2001;Bhagwat, 2003;Koo and Jaykus, 2003;Somer and Kashi, 2003;D'Agostino et al, 2004;Rodriguez-Lazaro et al, 2004b;Wang et al, 2004;Rudi et al, 2005). Many are based on the detection of virulence genes in L. monocytogenes and have been tested in a variety of foods.…”

Outbreak of encephalitic listeriosis in red-legged partridges (Alectoris rufa)

“…monocytogenes was identified using an in-house duplex real-time PCR exonuclease-assay (LM-PCR) amplifying simultaneously a specific 112 bp fragment of the L. monocytogenes haemolysin gene and a specific 80 bp fragment of the L. monocytogenes phospholipase A gene (Nogva et al, 2000). Each reaction contained 25 μl of 1 × TaqMan™ Fast Universal PCR Master Mix (Applied Biosystems, Life Technologies, Carlsbad, CA, USA), 0.3 μM of each of the forward and reverse primers (Eurogentec, Seraing, Belgium), 0.1 μM of each fluorescently labelled probes (Eurogentec) and 5 μl of crude or purified DNA extract.…”

Application of SYBR Green I and TaqMan probe-based real-time PCRs for the identification of Listeria spp. and Listeria monocytogenes

“…The reaction was performed in a 25 µL reaction mixture containing the appropriate concentration of each primer and 5 µL of DNA. One pair of primers detecting the 23S rDNA gene of the genus Listeria (19) and two pairs of primers amplifying the hlyA gene of L. monocytogenes (10,15) were selected (Table 1) and synthesised by Genomed (Poland). Primer concentrations (0.1 µM, 0.25 µM, 0.5 µM, 1.0 µM, 1.5 µM, and 2.0 µM) were evaluated to optimise the reaction.…”

“…The reaction was performed in a 25 µL reaction mixture containing the appropriate concentration of each primer and probe, and 5 µL of DNA. Based on the literature data, the sequences of three different pairs of primers and probes were selected (10,15,19). The characteristics of the primers and probes for real-time PCRs are shown in Tables 1 and 2.…”