Enterohemorrhagic E.coli 0157:H7 and other EHEC have emerged as important food borne pathogens. Rapid methods to diagnose these bacteria and to study their epidemiology are needed. Although gel electrophoretic typing of PCR-amplified DNA is a sensitive method for detecting microbial pathogens, it is time-consuming and difficult to automate. Our company has developed a gel-free method for genetic fingerprinting, DNA MTase Genotyping, which can be used to diagnose viral or bacterial pathogens in a few minutes. Analysis relies upon three steps: [i] PCR amplification of short (approximately 60 to 80 b.p.) DNA fragments using a biotinylated primer, [ii] Sequence-specific methylation with 3H-methyl- SAM, [iii] Capture and detection of 3H-methyl-DNA using Streptavidin-SPA beads. After PCR amplification and 3H-methylation, detection is achieved by solid phase Scintillation Proximity Assay (SPA). Because this method requires no electrophoresis and minimal liquid handling or pipetting, it can be readily automated. Phase l Research is proposed to use rapid DNA MTase Genotyping and SPA detection to diagnose pathogenic bacteria, using E.coli 0157:H7 as an instructive model. Ordered maps of restriction-modification sites in uidA, Stx1, and Stx2 genes will be constructed from PCR-amplified EHEC bacterial DNA in approximately 30 minutes. PROPOSED COMMERCIAL APPLICATIONS: Commercial products include: diagnostic test kits for bacterial pathogens; MTase enzymes, manufacture/licensing of DNA MTase Genotyping kits (primers, enzymes, 3H-methyl-SAM, and SPA beads); automated instruments and reagents for ordered gene mapping.
