The Tadpole Assay Multuplex Platform for Biodefense Agent Diagnostics

用于检测生物防御病原体的诊断试剂和测定是公共安全的关键需求。两个关键 用于成功的分子诊断测定的组分是直接结合到细胞的传感器组分。 靶向生物体或由生物体分泌的产物的信号结构域，以及指示， 当传感器已经结合靶分子时，灵敏度。在过去的18个月里，我们实施了 成熟的技术：噬菌体展示，共价蛋白质-DNA连接，实时PCR，沿着结果 从我们的抗原发现研究，开始创建一个强大的诊断测定，以检测是否存在 生物和环境样品中的土拉热弗朗西丝菌（Ft）以及针对 针对FT。这些检测的核心蛋白质-DNA嵌合体被称为蝌蚪，它们能够 与类似的酶联免疫检测相比， 免疫吸附测定（ELISA）。此外，这些诊断能够识别靶分子， 浓度动态范围宽。 在本提案中，我们将对蝌蚪试验进行多重检测，以包括额外的发热和腹泻因子， 在总体WRCE诊断主题计划中概述。裂谷热病毒和隐孢子虫 将作为平台WRCE诊断方法和代理列表的初始比较对照 将在未来几年根据WRCE计划进行扩展。此外，我们将努力整合 在WRCE诊断小组中正在开发的其他平台的基本技术，如横向 流动微流体，以扩大蝌蚪诊断的效用，因为它们进一步多路复用， 并且除了它们的用途之外还适于护理点使用 在参考实验室。

Diagnostic reagents and assays to detect biodefense pathogens are critical needs for public safety. Two key components for a successful molecular diagnostic assay are a sensor component that binds directly to the targeted organism, or to a product secreted by the organism, and a signal domain that indicates, with great sensitivity, when the sensor has bound the target molecule. Over the past 18 months we have implemented proven technologies: phage-display, covalent protein-DNA linkage, and real-time PCR, along with the results from our antigen discovery research, to begin to create a powerful diagnostic assay to detect the presence of Francisella tularensis (Ft) in biological and environmental samples as well as immune responses directed against Ft. The protein-DNA chimeras central to these assays are called tadpoles, which are capable of achieving a much greater level of sensitivity (~106-fold greater) compared to analogous enzyme-linked immunosorbent assays (ELISAs). Furthermore these diagnostics are able to identify target molecules over a wide dynamic range of concentrations. In this proposal, we will multiplex the tadpole assay to include additional fever and diarrheal agents as outlined in the overall WRCE diagnostic theme plan. Ft, Rift Valley fever virus, and Cryptosporidium parvum will serve as initial, comparative controls for the platform WRCE diagnostic approaches, and the agent list will be expanded in later years according to the WRCE plan. Furthermore, we will seek to integrate elemental technologies from other platforms being developed in the WRCE diagnostic group, such as lateral flow microfluidics, to expand the utility of tadpole diagnostics as they are further multiplexed for the simultaneous detection of numerous agents and are adapted for point-of-care usage in addition to their use in the reference laboratory.