Improved Pathogen Detection by Fluorescence Correlation

描述（由申请人提供）：鉴于最近的全球事件，生物战制剂构成的威胁越来越受到关注。生物战病原体感染的许多早期症状与常见疾病难以区分。因此，在接触后不久进行早期发现感染的快速诊断分析是加快治疗和减少感染传播的必要步骤。目前的快速检测技术利用DNA纯化扩增（PCR）或特异性抗体检测（ELISA），因此需要不同的检测仪器。

DESCRIPTION (provided by applicant): 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.