The threats posed by agents of biological warfare have gained increasing attention in light of recent global events. Many early symptoms of infection by agents of biological warfare are indistinguishable from commonly observed diseases. For this reason, rapid, diagnostic assays for early detection of infection, soon after exposure, are an essential step to speed treatment and reduce the spread of infection. Current rapid assay technologies make use of DNA purification and amplification (PCR) or the specificity of antibody detection (ELISA) and therefore require different detection instruments. In this proposal, we describe development of probe-set technology that can be used on a single FCS Diagnostic Instrument that offers rapid, specific detection of pathogens using probe-sets that target DNA, protein antigens, or both simultaneously. FCS is a single-particle detection method that measures fluctuations in fluorescence intensity in a small volume. Our preliminary studies show that we can detect a single bacterium in a small sample volume when labeled with a membrane specific probe. The focus of this proposal is to utilize FCS in a mode which crosscorrelates multiple molecular targets, both protein antigens and specific nucleic acid sequences, to effect diagnostic screens that can be used for early, and definitive diagnosis of disease. FCS offers two distinct advantages over current rapid assay technologies. First, a single instrument can be used to assay for a combination of probes. This enables diagnostic laboratories to take advantage of the best available probe targets without implementing different assay types on different instruments. The second advantage comes from crosscorrelation of multiple probes. This approach improves the probability of detecting true-positive results, and reduces the observation of false-positives. Although development of probe-sets are optimized for FCS diagnostics, the systematic approach employed and the production of specific probes can be applied to other agents of biological warfare with the same instrumentation.
Thesaurus Terms: biohazard detection, diagnostic test, fluorescence spectrometry, fluorescent dye /probe, technology /technique development Bacillus anthracis, antibody, bioterrorism /chemical warfare, nucleic acid purification, protein enzyme linked immunosorbent assay, polymerase chain reaction
