The Tadpole Assay Multuplex Platform for Biodefense Agent Diagnostics

检测生物防御性病原体的诊断试剂和检测方法是公共安全的关键需求。两把钥匙 成功的分子诊断分析的组件是直接结合到 靶向有机体，或由有机体分泌的产物，以及一个信号结构域，以巨大的 敏感度，当传感器与目标分子结合时。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连接和实时聚合酶链式反应，以及结果 从我们的抗原发现研究开始创建一种强大的诊断分析来检测是否存在 生物和环境样品中的土拉氏弗朗西斯杆菌及其定向免疫反应 对阵堡垒。这些检测的核心蛋白质-DNA嵌合体被称为蝌蚪，它们能够 与类似的酶联反应相比，实现了更高的敏感度(~106倍 免疫吸附试验(ELISA)。此外，这些诊断方法能够通过 浓度动态范围宽。 在这项建议中，我们将多重蝌蚪化验，以包括额外的发烧和腹泻病原体 在整个WRCE诊断主题计划中概述。FT、裂谷热病毒和微小隐孢子虫 将作为平台WRCE诊断方法和代理名单的初始比较控制 根据WRCE计划，将在未来几年扩大。此外，我们将寻求整合 来自WRCE诊断组正在开发的其他平台的基本技术，如横向 流动微流体学，以扩大蝌蚪诊断的用途，因为它们被进一步多路传输到 同时检测多种试剂，除使用外，还适用于护理点使用 在参考实验室里。

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.