Improved Pathogen Detection by Fluorescence Correlation

描述(申请人提供)：鉴于最近的全球事件，生物战毒剂构成的威胁越来越受到关注。生物战制剂感染的许多早期症状与通常观察到的疾病难以区分。因此，在暴露后不久进行快速诊断化验以及早发现感染，是加快治疗和减少感染传播的重要步骤。目前的快速检测技术利用DNA纯化和扩增(PCR)或抗体检测的特异性(ELISA)，因此需要不同的检测仪器。 在本提案中，我们描述了可用于单个FCS诊断仪器的探针组技术的发展，该仪器使用针对DNA、蛋白质抗原或同时针对DNA和蛋白质抗原的探针组提供对病原体的快速、特异检测。FCS是一种单颗粒检测方法，它测量小体积内荧光强度的波动。我们的初步研究表明，当用膜特异性探针标记时，我们可以在小样本体积中检测到单个细菌。这项建议的重点是以一种使多个分子靶标相互关联的模式利用FCS，包括蛋白质抗原和特定的核酸序列，以实现可用于疾病的早期和明确诊断的诊断筛选。 与目前的快速检测技术相比，FCS有两个明显的优势。首先，一台仪器可以用来检测多种探针的组合。这使得诊断实验室能够利用最好的可用探针目标，而无需在不同的仪器上实施不同的分析类型。第二个优势来自多个探针的互相关性。这种方法提高了检测到真阳性结果的概率，并减少了假阳性的观察。虽然探针组的开发是针对FCS诊断进行优化的，但所采用的系统方法和特定探头的生产也可以应用于使用相同仪器的其他生物战制剂。

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.