Quantification detection of Listeria monocytogenes and Listeria innocua by real-time PCR: assessment of hly, iap, and lin02483 targets and AmpliFluor technology

• Published in: Applied and environmental microbiology Vol. 70; no. 3; pp. 1366 - 1377
• Main Authors: Rodriguez-Lazaro, D, Hernandez, M, Scortti, M, Esteve, T, Vazquez-Boland, J.A, Pla, M
• Format: Journal Article
• Language: English
• Published: 2004
• Subjects: Ampifluor technology; bacterial proteins; DNA primers; fluorescence; food microbiology; food pathogens; genes; genetic polymorphism; hemolysins; hly gene; iap gene; invasion-associated surface protein p60; lin02483 gene; Listeria innocua; Listeria monocytogenes; listeriolysin O; microbial detection; molecular sequence data; nucleotide sequences; polymerase chain reaction
• ISSN: 0099-2240

We developed and assessed real-time PCR (RTi-PCR) assays for the detection and quantification of the food-borne pathogen Listeria monocytogenes and the closely related nonpathogenic species L. innocua. The target genes were hly and iap for L. monocytogenes and lin02483 for L. innocua. The assays were 100% specific, as determined with 100 Listeria strains and 45 non-Listeria strains, and highly sensitive, with detection limits of one target molecule in 11 to 56% of the reactions with purified DNA and 3 CFU in 56 to 89% of the reactions with bacterial suspensions. Quantification was possible over a 5-log dynamic range, with a limit of 15 target molecules and R2 values of >0.996. There was an excellent correspondence between the predicted and the actual numbers of CFU in the samples (deviations of <23%). The hly-based assay accurately quantified L. monocytogenes in all of the samples tested. The iap-based assay, in contrast, was unsuitable for quantification purposes, underestimating the bacterial counts by 3 to 4 log units in a significant proportion of the samples due to serovar-related target sequence variability. The combination of the two assays enabled us to classify L. monocytogenes isolates into one of the two major phylogenetic divisions of the species, I and II. We also assessed the new AmpliFluor technology for the quantitative detection of L. monocytogenes by RTi-PCR. The performance of this system was similar to that of the TaqMan system, although the former system was slightly less sensitive (detection limit of 15 molecules in 45% of the reactions) and had a higher quantification limit (60 molecules).