A validated PCR-based method to detect Listeria monocytogenes using raw milk as a food model - Towards an international standard

M. D'Agostino, M. Wagner, J.A. Vazquez-Boland, T. Kuchta, R. Karpiskova, Jeffrey Hoorfar, S. Novella, M. Scortti, J. Ellison, A. Murray, I. Fernandes, M. Kuhn, J. Pazlarova, A. Heuvelink, N. Cook

Research output: Contribution to journalJournal articlepeer-review

Abstract

A PCR assay with an internal amplification control was developed for Listeria monocytogenes. The assay has a 99% detection probability of seven cells per reaction. When tested against 38 L. monocytogenes strains and 52 nontarget strains, the PCR assay was 100% inclusive (positive signal from target) and 100% exclusive (no positive signal from nontarget). The assay was then evaluated in a collaborative trial involving 12 European laboratories, where it was tested against an additional 14 target and 14 nontarget strains. In that trial, the inclusivity was 100% and the exclusivity was 99.4%, and both the accordance (repeatability) and the concordance (reproducibility) were 99.4%. The assay was incorporated within a method for the detection of L. monocytogenes in raw milk, which involves 24 It of enrichment in half-Fraser broth followed by 16 h of enrichment in a medium that can be added directly into the PCR. The performance characteristics of the PCR-based method were evaluated in a collaborative trial involving 13 European laboratories. In that trial, a specificity value (percentage of correct identification of blank samples) of 81.8% was obtained; the accordance was 87.9%, and the concordance was 68.1%. The sensitivity (correct identification of milk samples inoculated with 20 to 200 L. monocytogenes cells per 25 ml) was 89.4%, the accordance was 81.2%, and the concordance was 80.7%. This method provides a basis for the application of routine PCR-based analysis to dairy products and other foodstuffs and should be appropriate for international standardization.
Original languageEnglish
JournalJournal of Food Protection
Volume67
Issue number8
Pages (from-to)1646-1655
ISSN0362-028X
Publication statusPublished - 2004

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