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Applied and Environmental Microbiology, December 2005, p. 9008-9012, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.9008-9012.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.

SHORT REPORT

A Novel Real-Time PCR for Listeria monocytogenes That Monitors Analytical Performance via an Internal Amplification Control

David Rodríguez-Lázaro,1,2* Maria Pla,1 Mariela Scortti,2,3 Héctor J. Monzó,2,3 and José A. Vázquez-Boland2,3

Institute of Food and Agricultural Technology (INTEA), University of Girona, Girona, Spain,1 Bacterial Molecular Pathogenesis Group, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford, United Kingdom,2 Facultad de Veterinaria, Universidad de León, León, Spain3

Received 25 February 2005/ Accepted 15 August 2005

We describe a novel quantitative real-time (Q)-PCR assay for Listeria monocytogenes based on the coamplification of a target hly gene fragment and an internal amplification control (IAC). The IAC is a chimeric double-stranded DNA containing a fragment of the rapeseed BnACCg8 gene flanked by the hly-specific target sequences. This IAC is detected using a second TaqMan probe labeled with a different fluorophore, enabling the simultaneous monitoring of the hly and IAC signals. The hly-IAC assay had a specificity and sensitivity of 100%, as assessed using 49 L. monocytogenes isolates of different serotypes and 96 strains of nontarget bacteria, including 51 Listeria isolates. The detection and quantification limits were 8 and 30 genome equivalents, and the coefficients for PCR linearity (R2) and efficiency (E) were 0.997 and 0.80, respectively. We tested the performance of the hly-IAC Q-PCR assay using various broth media and food matrices. Fraser and half-Fraser media, raw pork, and raw or cold-smoked salmon were strongly PCR-inhibitory. This Q-PCR assay for L. monocytogenes, the first incorporating an IAC to be described for quantitative detection of a food-borne pathogen, is a simple and robust tool facilitating the identification of false negatives or underestimations of contamination loads due to PCR failure.

* Corresponding author. Mailing address: Veterinary Molecular Microbiology Section, Faculty of Medical and Veterinary Sciences, University of Bristol, Langford BS40 5DU, United Kingdom. Phone: 44 117 928 9667. Fax: 44 117 928 9505. E-mail: david.rodriguez{at}bris.ac.uk.

Applied and Environmental Microbiology, December 2005, p. 9008-9012, Vol. 71, No. 12
0099-2240/05/$08.00+0     doi:10.1128/AEM.71.12.9008-9012.2005
Copyright © 2005, American Society for Microbiology. All Rights Reserved.



This article has been cited by other articles:

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