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Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set.

ABSTRACT: 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 10(3) and 10(4) 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.

SUBMITTER: Koo K

PROVIDER: S-EPMC143584 | biostudies-literature | 2003 Feb

REPOSITORIES: biostudies-literature

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Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set.

Koo Kai K Jaykus Lee-Ann LA

Applied and environmental microbiology 20030201 2

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 monocytog ...[more]

PMID: 12571032

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Project description:BACKGROUND:Listeria monocytogenes is a widespread foodborne pathogen that can cause listeriosis, a potentially fatal infection. L. monocytogenes is subdivided into four phylogenetic lineages, with the highest incidence of listeriosis occurring within lineage I followed by lineage II. Strains of L. monocytogenes differ in their phenotypic characteristics, including virulence. However, the genetic bases for these observed differences are not well understood, and current efforts to monitor L. monocytogenes in food consider all strains to be equally virulent. We use a comparative genomics approach to identify genes and single nucleotide polymorphisms (SNPs) in 174 clinical and food isolates of L. monocytogenes that potentially contribute to virulence or the capacity to adapt to food environments. RESULTS:No SNPs are significantly associated with food or clinical isolates. No genes are significantly associated with food or clinical isolates from lineage I, but eight genes consisting of multiple homologues are associated with lineage II food isolates. These include three genes which encode hypothetical proteins, the cadmium resistance genes cadA and cadC, the multi-drug resistance gene ebrB, a quaternary ammonium compound resistance gene qac, and a regulatory gene. All eight genes are plasmid-borne, and most closed L. monocytogenes plasmids carry at least five of the genes (24/27). In addition, plasmids are more frequently associated with lineage II food isolates than with lineage II clinical isolates. CONCLUSIONS:We identify eight genes that are significantly associated with food isolates in lineage II. Interestingly, the eight genes are virtually absent in lineage II outbreak isolates, are composed of homologues which show a nonrandom distribution among lineage I serotypes, and the sequences are highly conserved across 27 closed Listeria plasmids. The functions of these genes should be explored further and will contribute to our understanding of how L. monocytogenes adapts to the host and food environments. Moreover, these genes may also be useful as markers for risk assessment models of either pathogenicity or the ability to proliferate in food and the food processing environment.

Dataset Information

Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set.

Publications

Detection of Listeria monocytogenes from a model food by fluorescence resonance energy transfer-based PCR with an asymmetric fluorogenic probe set.

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