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Bacterial detection in foods by nucleic acid probes is limited by microflora competition during selective enrichment. Probe target concentration by extraction and fractionation of enrichments may diminish this limitation. The 1-h AccuProbe chemiluminescent culture identification test for Listeria monocytogenes was used as a model. Its high detection threshold provides a stringent challenge for evaluating enrichmentwork-up protocols. Detection of L. monocytogenes, at 14 colony-forming units/g food,was not consistently possible in 48 h enrichment cultures usingAccuProbe. Concentration by cell sedimentationwas occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sedimentswere sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNAwas separated from non-RNA material by extraction with phenol and precipitation with ethanol. Enrichments (250 mL) were concentrated 2500-fold, and the limitation was food RNA volume. A strongly competitive Enterococcus faecium food isolate was used to demonstrate the effect of artificial competition on the kit's ability to detect L. monocytogenes in enrichments. High competitor concentrations repressed the level of the target below the detection threshold, but concentration of r-RNA enabled detection of L. monocytogenes. The effectiveness of this enrichment sample work-up was demonstrated with naturally contaminated hummus.

Improved DNA Probe Detection of Listeria monocytogenes in Enrichment Culture After Physical-Chemical Fractionation