Improved Pathogen Detection by Fluorescence Correlation

描述（由申请人提供）：鉴于最近的全球事件，生物战剂构成的威胁日益受到关注。生物战剂感染的许多早期症状与常见疾病没有区别。因此，在暴露后不久进行快速诊断检测以及早发现感染，是加速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 (ELISA) 的特异性，因此需要不同的检测仪器。 在本提案中，我们描述了探针组技术的开发，该技术可用于单个 FCS 诊断仪器，使用同时靶向 DNA、蛋白质抗原或两者的探针组提供快速、特异性的病原体检测。 FCS是一种单粒子检测方法，可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，我们可以检测小体积样品中的单个细菌。该提案的重点是以交叉关联多个分子靶标（蛋白质抗原和特定核酸序列）的模式利用 FCS 来实现可用于疾病早期和明确诊断的诊断筛选。 与当前的快速检测技术相比，FCS 具有两个明显的优势。首先，可以使用单个仪器来测定探针组合。这使得诊断实验室能够利用最佳的可用探针目标，而无需在不同的仪器上实施不同的检测类型。第二个优点来自多个探针的互相关。这种方法提高了检测真阳性结果的概率，并减少了假阳性的观察。尽管探针组的开发针对 FCS 诊断进行了优化，但所采用的系统方法和特定探针的生产可以应用于具有相同仪器的其他生物战剂。

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.