Improved DNA probe detection of Listeria monocytogenes in enrichment culture after physical-chemical fractionation

• Published in: Journal of AOAC International 2006, Vol.89 (1), p.172-179
• Main Authors: Duvall, R.E, Eklund, M, Tran, T.T, Hitchins, A.D
• Format: Article
• Language: English
• Subjects: bacterial contamination; Bacteriological Techniques; Binding, Competitive; Biological and medical sciences; Chemistry Techniques, Analytical - methods; Colony Count, Microbial; Culture Media; DNA primers; DNA probes; DNA Probes - chemistry; DNA Probes - pharmacology; enrichment culture; Enterococcus faecium - metabolism; Ethanol - chemistry; Food Analysis; food contamination; Food industries; Food Microbiology; food pathogens; Fundamental and applied biological sciences. Psychology; General aspects; Identification and classification; Listeria monocytogenes; Listeria monocytogenes - metabolism; Methods of analysis, processing and quality control, regulation, standards; microbial detection; Phenol - chemistry; rapid methods; RNA - analysis; RNA - chemistry; selective media; Stem Cells; Temperature; Time Factors
• ISSN: 1060-3271; 1944-7922
• DOI: 10.1093/jaoac/89.1.172

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 enrichment work-up protocols. Detection of L. monocytogenes, at 1-4 colony-forming units/g food, was not consistently possible in 48 h enrichment cultures using AccuProbe. Concentration by cell sedimentation was occasionally helpful but the volume of co-sedimented food limited concentration to about 10-fold. To improve concentration, enrichment sediments were sonicated or enzymatically lysed to release the probe's target, r-RNA. The RNA was 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.