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Applied and Environmental Microbiology, March 2004, p. 1366-1377, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1366-1377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.

Quantitative Detection of Listeria monocytogenes and Listeria innocua by Real-Time PCR: Assessment of hly, iap, and lin02483 Targets and AmpliFluor Technology

David Rodríguez-Lázaro,1 Marta Hernández,2 Mariela Scortti,3 Teresa Esteve,2 José A. Vázquez-Boland,3 and Maria Pla1,2*

Institute of Food and Agricultural Technology, University of Girona, Girona,1 Institute of Molecular Biology-Consejo Superior de Investigaciones Científicas, Barcelona, Spain,2 Veterinary Molecular Microbiology Section, University of Bristol, Langford BS40 5DU, United Kingdom3

Received 21 August 2003/ Accepted 21 November 2003

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).

* Corresponding author. Mailing address: Institute of Food and Agricultural Technology, University of Girona, Campus Montilivi s/n, E-17071 Girona, Spain. Phone: 34-972 419852. Fax: 34-972 418399. E-mail: maria.pla{at}udg.es.

Applied and Environmental Microbiology, March 2004, p. 1366-1377, Vol. 70, No. 3
0099-2240/04/$08.00+0     DOI: 10.1128/AEM.70.3.1366-1377.2004
Copyright © 2004, American Society for Microbiology. All Rights Reserved.



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