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Phage-mediated bioluminescent detection of Yersinia pestis.

噬菌体介导的鼠疫耶尔森菌生物发光检测。

基本信息

批准号：
7664751
负责人：
DAVID A SCHOFIELD
金额：
$ 11.08万
依托单位：
GUILD ASSOCIATES, INC.
依托单位国家：
美国
项目类别：
财政年份：
2009
资助国家：
美国
起止时间：
2009-03-12 至 2009-08-31
项目状态：
已结题

来源：
https://reporter.nih.gov/project-details/7664751
关键词：
Agar Agar Gel Electrophoresis Applied Research Attenuated Bacillus anthracis Bacteriophage Typing Bacteriophages Biological Assay Bioluminescence Bubonic Plague Categories Cell Count Centers for Disease Control and Prevention (U.S.)Cessation of life Characteristics Clinical Cytolysis Detection Development Diagnosis Diagnostic Diagnostic tests Disease Disease Outbreaks Dose Early Diagnosis Effectiveness Encapsulated Ensure Environment Feasibility Studies Foundations Functional RNA Future Generations Genes Genome Goals Growth Health Professional Human Laboratories Lead Life Light Lytic Measures Mediating Medical Methodology National Institute of Allergy and Infectious Disease Phase Phase I Clinical Trials Plague Play Pneumonic Plague Process Public Health Reaction Time Recombinants Recording of previous events Reporter Research Role Sampling Signal Transduction Strategic Planning System Technology Temperature World Health Organization Yersinia pestis aerosolized base biodefense commercial application fitness homologous recombination mortality novel pathogen prevent public health relevance rapid detection response tool

项目摘要

DESCRIPTION (provided by applicant): Yersinia pestis is designated by the Centers for Disease Control (CDC) and NIAID as a Category A priority bacterial pathogen. Y. pestis is the etiological agent of the plague (Black Death), a transmissible disease that has been responsible for millions of deaths throughout the course of history. Although the natural occurrence of this disease is now relatively rare, the possibility of terrorist groups using Y. pestis as a bioweapon is real. Because of the disease's inherent communicability, rapid clinical course, and high mortality, it is critical that an outbreak be detected quickly. Therefore methodologies that provide rapid detection and diagnosis are essential to ensure immediate implementation of public health measures and activation of crisis management. The long-term goal and commercial application of this application is to develop a plague diagnostic detection kit. The objective of this Phase I research is to generate a luxAB ('light')-tagged phage that can specifically detect Y. pestis. Y. pestis specific phages are currently used by the CDC and the World Health Organization (WHO) as a diagnostic standard for the confirmed identification of Y. pestis; however, the phage lysis assays are laboratory based and require 18-24 h to complete. In contrast, the technology described in this application will not require sample processing, a laboratory environment, or extensive incubation periods. Specific Aim 1 will generate a Y. pestis reporter phage by integrating the bacterial luxAB genes into a non-coding region within the genome of the plague diagnostic phage. Recombinant luxAB-tagged phage will be identified and isolated based on the ability of infected cultures to emit bioluminescence. Specific Aim 2 will perform feasibility studies to demonstrate that the luxAB-phage can effectively be used as a Y. pestis detection system. An attenuated Y. pestis strain (exempt select agent, BSL2 pathogen) will be used. The sensitivity limits of detection, the signal response time, and the dose-response characteristics will be determined. Collectively, this application will generate the proof of principal studies for a novel 'light producing' reporter phage that rapidly and specifically detects Y. pestis. Our preliminary results have demonstrated the feasibility of a similar based approach for the detection of non-encapsulated Bacillus anthracis strains. Since a future direction of NIAID applied research is to "encourage development of multiplex diagnostics" for Category A pathogens (2007 NIAID Strategic Plan for Biodefense Research), one of the long-term goals of Guild Associates is to develop a reporter phage cocktail that can be used for the diagnostic identification of different Category A bacterial pathogens, such as B. anthracis and Y. pestis. PUBLIC HEALTH RELEVANCE: This application is significant because it will generate the proof of principle results for a rapid plague diagnostic test kit. A rapid diagnostic test kit is essential in order to quickly implement public health measures which will save lives and prevent the spread of the disease.

描述（由申请人提供）：鼠疫耶尔森菌被疾病控制中心（CDC）和NIAID指定为A类优先细菌病原体。Y.鼠疫是鼠疫（黑死病）的病原体，黑死病是一种传染病，在整个历史过程中造成数百万人死亡。尽管这种疾病的自然发生现在相对罕见，但恐怖组织利用Y.鼠疫作为生物武器是真实的。由于该疾病固有的传染性、快速的临床病程和高死亡率，迅速发现疫情至关重要。因此，提供快速检测和诊断的方法对于确保立即执行公共卫生措施和启动危机管理至关重要。本申请的长期目标和商业应用是开发鼠疫诊断检测试剂盒。第一阶段研究的目标是产生一种可以特异性检测Y染色体的luxAB（“light”）标记的噬菌体。鼠疫Y.目前，疾病预防控制中心和世界卫生组织（WHO）使用鼠疫特异性噬菌体作为确诊Y.鼠疫杆菌;然而，噬菌体裂解测定是基于实验室的，需要18-24小时才能完成。相比之下，本申请中描述的技术将不需要样品处理、实验室环境或长时间的孵育期。具体目标1将生成Y。通过将细菌luxAB基因整合到鼠疫诊断噬菌体的基因组内的非编码区中，将基于感染的培养物发射生物发光的能力来鉴定和分离重组luxAB标记的噬菌体。Specific Aim 2将进行可行性研究，以证明luxAB-噬菌体可有效地用作Y。鼠疫检测系统减毒Y.将使用鼠疫菌株（豁免选择剂，BSL 2病原体）。将确定检测的灵敏度限值、信号响应时间和剂量响应特性。总的来说，这一应用将产生一种新的“发光”报告噬菌体，快速和特异性检测Y的主要研究的证据。鼠疫我们的初步研究结果表明，一个类似的基础上的方法用于检测非封装炭疽芽孢杆菌菌株的可行性。由于NIAID应用研究的未来方向是“鼓励开发A类病原体的多重诊断”（2007 NIAID生物防御研究战略计划），Guild Associates的长期目标之一是开发可用于诊断鉴定不同A类细菌病原体（如B）的报告噬菌体混合物。炭疽病和Y.鼠疫公共卫生相关性：该应用是重要的，因为它将产生用于快速鼠疫诊断检测试剂盒的原理证明结果。快速诊断检测包对于迅速执行公共卫生措施，拯救生命和防止疾病传播至关重要。