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Applied and Environmental Microbiology, February 2003, p. 1082-1088, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1082-1088.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.

Detection of Listeria monocytogenes from a Model Food by Fluorescence Resonance Energy Transfer-Based PCR with an Asymmetric Fluorogenic Probe Set{dagger}

Kai Koo and Lee-Ann Jaykus*

Department of Food Science, College of Life Science and Agriculture, North Carolina State University, and Southeast Dairy Foods Research Center, Raleigh, North Carolina 27695-7624

Received 20 March 2002/ Accepted 1 November 2002

It has been shown that fluorescence resonance energy transfer (FRET)-based PCR, including the TaqMan assay and molecular beacons, has potential for rapid detection of pathogens. In these promising real-time detection assays a single internal oligonucleotide probe labeled on both the 5' (reporter) and 3' (quencher) ends is used for selective generation of fluorescence. In this paper, we describe the use of a previously reported novel probe design for FRET-based PCR detection of Listeria monocytogenes in pure culture and in a model food commodity. In the assay described here an asymmetric probe set is used; this probe set consists of a long 5' fluorescein-labeled reporter probe and a short, complementary 3' DABCYL-labeled quencher oligonucleotide, which are used in a 5' nuclease amplification and detection assay. By using the listeriolysin O (hly) and p60 (iap) genes as amplification targets, the performance of two primer-probe sets in amplification and subsequent detection of target DNA was evaluated. In studies performed with pure cultures of L. monocytogenes, the PCR profiles indicated that the relative change in fluorescence intensity was correlated with both the initial number of cells and the accumulation of specific amplicons for both hly and iap gene fragments. Experiments were also done to determine the applicability of the method to the detection of L. monocytogenes by targeting hly DNA and its short-lived mRNA product in a model food commodity. Twenty-five-milliliter samples of reconstituted nonfat dry milk (NFDM) were seeded with L. monocytogenes and processed to concentrate the bacteria by centrifugation, and this was followed by nucleic acid extraction and amplification with hly-specific primers. Endpoint detection of PCR and reverse transcription-PCR amplicons could be achieved at inoculum levels of 103 and 104 CFU of L. monocytogenes/25 ml of NFDM, respectively. This study demonstrated that this asymmetric FRET-based amplification and detection protocol provides an alternative approach for endpoint detection of nucleic acid amplification products as applied to detection of pathogens in a model food.

* Corresponding author. Mailing address: Department of Food Science, North Carolina State University, Raleigh, NC 27695-7624. Phone: (919) 513-2074. Fax: (919) 515-7124. E-mail: leeann_jaykus{at}ncsu.edu.

{dagger} Paper no. FSR 02-14 of the Journal Series of the Department of Food Science, North Carolina State University, Raleigh.

Applied and Environmental Microbiology, February 2003, p. 1082-1088, Vol. 69, No. 2
0099-2240/03/$08.00+0     DOI: 10.1128/AEM.69.2.1082-1088.2003
Copyright © 2003, American Society for Microbiology. All Rights Reserved.



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