WSU ZRU Listeria monocytogenes subtyping project

We have been developing genomic microarrays to characterize several bacterial species including Listeria monocytogenes. The goal of the present proposal is to migrate phylogenetically informative probe sequences from our microarrays to a bead array format that permits rapid subtyping of L. monocytogenes isolates. Listeria subtyping probes are used to develop oligonucleotides probes that are subsequently conjugated to microsphere beads. Each unique probe sequence is attached to a uniquely colored bead. Genomic DNA is extracted from cultured bacteria and labeled using nick translation followed by hybridization to a collection of beads. After hybridization and secondary labeling of genomic DNA with a fluorophore, the beads are counted using a flow-cytometer where one laser assesses each bead color (i.e., probe sequence) and a second laser determines if there is genomic DNA hybridized to the bead. This system can accommodate 96-well plates. We plan to use this assay system to compare and map the distribution of L. monocytogenes subtypes collected from a variety of sources throughout North America.

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Food and Waterborne Diseases Integrated Research Network

National Institute of Allergy and Infectious Diseases 6610 Rockledge Drive, MSC 6612 Bethesda, MD 20892-6612

National Institutes of Health (NIH) 9000 Rockville Pike Bethesda, Maryland 20892

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