Rapid, Potentially Automatable, Method Extract Biomarkers for HPLC/ESI/MS/MS to Detect and Identify BW Agents

Our program proposes to concentrate on the rapid recovery of signature biomarkers based on automated high-pressure, high-temperature solvent extraction ASE andor supercritical fluid extraction SFE to produce lipids, nucleic acids and proteins sequentially concentrated and purified in minutes with yields especially from microeukaryotes, Gram-positive bacteria and spores. Lipids are extracted in higher proportions greater than classical one-phase, room temperature solvent extraction without major changes in lipid composition. High performance liquid chromatography HPLC with or without derivatization, electrospray ionization ESI and highly specific detection by mass spectrometry MS particularly with MSexp n provides the detection, identification and because the signature lipid biomarkers are both phenotypic as well as genotypic biomarkers, insights into potential infectivity of BW agents. Feasibility has been demonstrated with detection, identification, and determination of infectious potential of Cryptosporidium parvum at the sensitivity of a single oocyst which is unculturable in vitro and accurate identification and prediction, pathogenicity, and drug-resistance of Mycobacteria spp.