James B Day
U.S. Food and Drug Administration, USA
Title: Bio-Plex suspension array immuno-detection of Listeria monocytogenes from lettuce and spinach using virulence protein inducing charcoal-activated enrichment media
Biography
Biography: James B Day
Abstract
Statement of the problem: Listeria monocytogenes, the causative agent of Listeriosis in humans, is a Gram-positive bacterium that is contracted via the consumption of contaminated foods. Various leafy green vegetables, including lettuce and spinach, have been implicated in human Listeriosis cases. 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. Methodology: In this study, we utilize a novel enrichment media containing activated charcoal as the key ingredient that induces overexpression and secretion of L. monocytogenes virulence proteins. The Bio-Plex suspension array system, based on Luminex xMAP technology, can then be utilized to specifically detect accumulated L. monocytogenes virulence proteins via a magnetic bead-antibody complex. Iceburg lettuce and packaged ready-to-eat spinach were treated with L. monocytogenes and incubated in preenrichment broth (Buffered Listeria Enrichment Broth) followed by incubation in charcoal activated media. The supernatant fraction was TCA precipitated and L. monocytogenes lysteriolysin O (LLO) was collected using magnetic microspheres conjugated to LLO specific antibody. A newly develop antibody that exclusively recognize L. monocytogenes LLO was used as the biotin conjugated secondary antibody and analysis was conducted using the Bio-Plex 200 system. Findings: As few as 1 CFU/ g of L. monocytogenes was detected in both foods tested. Whole cell fractions from 14h activated charcoal enrichments were also analyzed using antibody that recognize both pathogenic and non-pathogenic Listeria species which also resulted in a detection limit of 1 CFU/ g. Internal control beads were also utilized to ensure proper instrumentation function, integrity of assay reagents and to eliminate the possibility of non-specific interactions. Conclusions and significance: This method is the first to specifically recognize and differentiate L. monocytogenes among other nonpathogenic Listeria species in various leafy greens using immune-detection.The total presumptive detection time can be achieved in less than 24h.