100A-22 |
Detection of low levels of pathogenic Listeria monocytogenes in 14 - 20 h using immunoseparation and cytotoxicity techniques |
K. M. NASCHANSKY, Department of Food Science, Purdue University, 1160 Food Science Building, West Lafayette, IN 47907 and A. K. Bhunia. The development of rapid detection methods for Listeria monocytogenes is crucial when considering that listeriosis targets immunocompromised individuals such as the elderly, pregnant women and young children and that conventional detection methods are not sufficient. While immunomagnetic separation has already been used for isolating Listeria cells from various types of samples, the limitations of lowered specificity, high cost and the lack of pathogenicity determination render this method insufficient. To overcome the limitations of current immunoseparation methods, we set out to develop a method using protein-A agarose beads (immunobeads) coated with a more specific antibody, testing both polyclonal and monoclonal, for L. monocytogenes that would not only increase the specificity and efficiency of the capture, but also decrease the overall cost of the immunoseparation method when compared to commercially-available magnetic beads for Listeria isolation. The immunobeads were allowed to capture Listeria cells from spiked or naturally contaminated hotdog samples following selective enrichment in half-Fraser broth for 0 - 24 h. The captured Listeria cells were tested for cytopathogenic action on a murine hybridoma B-lymphocyte, Ped-2E9 cell line by Trypan blue viability staining and an alkaline phosphatase-based cytotoxicity assay, producing results in 1 - 2 h. Detection and confirmation of pathogenicity of the captured L. monocytogenes with initial contamination levels of 1 - 100 CFU/100 ml from hotdog extracts was obtained in 14 - 20 h. The natural isolates were further confirmed to be CAMP positive (hemolytic) and genomic patterns were identical to L. monocytogenes as determined by Riboprinter® analysis. Through the use of our two-step rapid method, using immunoseparation and cytotoxicity assay, low levels (1 - 100 CFU) of L. monocytogenes could be isolated, detected and confirmed as pathogenic in just 14 - 20 h, saving food processors valuable time, while ensuring a safer food supply for the public.
Session 100A, Food Microbiology: General II
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