Bio-Plex suspension array immuno-detection of Listeria monocytogenes from cantaloupe and packaged salad using virulence protein inducing activated charcoal enrichment media.

Listeria monocytogenes, the causative agent of listeriosis in humans, is a Gram-positive bacterium that is contracted via the ingestion of contaminated foods. Two of the largest outbreaks of listeriosis occurred following consumption of tainted cantaloupe and packaged salads. Molecular methods and immuno-based techniques for detection of L. monocytogenes in these food matrices can be difficult due to the presence of assay inhibiting elements. In this study, we utilized a novel enrichment media containing activated charcoal as the key ingredient that induces hyperactive expression and secretion of L. monocytogenes virulence proteins. The Bio-Plex suspension array system, based on Luminex xMAP technology, was subsequently employed to specifically detect accumulated L. monocytogenes secreted and membrane bound proteins via paramagnetic microsphere-antibody complexes. Cantaloupe and packaged salad samples were treated with a dilution series of L. monocytogenes and incubated in activated charcoal media following a short pre-enrichment step in Buffered Listeria Enrichment Broth. Secreted L. monocytogenes lysteriolysin O was captured using magnetic microsphere-antibody conjugates and measured using the Bio-Ple×200 analyzer. As few as 10 CFU/g of L. monocytogenes was detected from both spiked cantaloupe and packaged salad samples. In addition, antibody conjugated microspheres targeting a membrane protein present on both pathogenic and nonpathogenic Listeria species was used to identify as few as 10 CFU/g of both pathogenic and nonpathogenic species in cantaloupe and packaged salad. This method presumptively identifies L. monocytogenes from cantaloupe and packaged salad in less than 24 h and non-pathogenic Listeria species within 22 h.