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

• 批准号: 8123772
• 负责人: DAVID A SCHOFIELD
• 金额: $ 39.53万
• 依托单位: GUILD ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2009
• 资助国家: 美国
• 起止时间: 2009-03-12 至 2013-02-28
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8123772
• 关键词: Animals; Attenuated; Bacteriophages; Biological Assay; Blood; Blood Circulation; Bubonic Plague; Categories; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Clinical; Cytolysis; Detection; Development; Diagnostic; Diagnostic tests; Disease; Disease Outbreaks; Early Diagnosis; Engineering; Ensure; Enterobacteriaceae; Environment; Exhibits; Fleas; Foundations; Genome; Geographic Locations; Goals; Grant; Growth; Health Professional; Human; Infection; Laboratories; Lead; Libraries; Life; Light; Lytic; Measures; Mediating; Methodology; Modeling; Mus; National Institute of Allergy and Infectious Disease; Pasteurella pseudotuberculosis; Patients; Phase; Phenotype; Plague; Play; Pneumonic Plague; Preparation; Process; Public Health; Recombinants; Recording of previous events; Reporter; Reporter Genes; Research; Sampling; Sensitivity and Specificity; Signal Transduction; Source; Specificity; Specimen; Sputum; Symptoms; System; Technology; Validation; Virulent; Vision; Work; World Health Organization; Yersinia; Yersinia pestis; absorption; aerosolized; aqueous; base; commercial application; disease diagnosis; mortality; mouse model; novel; pathogen; prevent; product development; rapid detection; rapid diagnosis; response; tool

DESCRIPTION (provided by applicant): Yersinia pestis is designated by the Centers for Disease Control (CDC) and NIAID as a Category A 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 proposal is to develop a plague diagnostic detection kit. The Phase I research successfully completed the proof of principle study and generated a recombinant luxAB ('light')-tagged reporter phage for the detection of Y. pestis. Y. pestis specific phage 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 2 to 3 days to complete. In contrast, the technology described in this proposal does not require sample processing, a laboratory environment, or extensive incubation periods. The Phase I results demonstrated that the luxAB reporter phage rapidly conferred a bioluminescent signal response to an attenuated Y. pestis strain and displayed potential as a plague diagnostic. The Phase II research will build upon the Phase I foundation with the goal of generating a plague diagnostic kit. The Phase II research will: (i) increase the specificity and sensitivity of the reporter phage for Y. pestis (Aim 1); (ii) demonstrate the utility of the reporter phage against a library of wild-type Y. pestis strains (Aim 2); (iii) analyze the specificity of the reporter phage for Y. pestis using a panel of pathogenic non-pestis Yersinia species (Aim 3), and (iv) analyze and optimize the ability of the detection system to function directly with clinical specimens (Aim 4). Collectively, this research will provide the foundation for a plague diagnostic kit using novel 'light producing' reporter phage that rapidly, sensitively, and specifically confer a bioluminescent phenotype to Y. pestis. The vision of the product is that it can be used as a diagnostic for suspected plague-infected patients with cultivated isolates or directly with infected clinical specimens in a laboratory setting or as a portable camera-based assay. The long-term goal of our research is to develop a reporter phage-based multiplex assay for the identification of different bacterial priority A pathogens. PUBLIC HEALTH RELEVANCE: This proposal is significant because it will generate a rapid plague diagnostic test kit. A rapid diagnostic test kit is essential in order to specifically diagnose the disease, prevent the spread of the disease, and to save lives.

描述(申请人提供)：鼠疫耶尔森氏菌被疾病控制中心(CDC)和NIAID指定为A类细菌病原体。鼠疫杆菌是鼠疫(黑死病)的病原体，黑死病是一种传染性疾病，在整个历史过程中造成了数百万人的死亡。虽然这种疾病的自然发生现在相对罕见，但恐怖组织使用鼠疫杆菌作为生物武器的可能性是真实存在的。由于这种疾病固有的传染性、快速的临床病程和高死亡率，迅速发现暴发是至关重要的。因此，提供快速检测和诊断的方法对于确保立即实施公共卫生措施和启动危机管理至关重要。这项提议的长期目标和商业应用是开发一种鼠疫诊断检测试剂盒。一期研究成功地完成了原理验证研究，获得了检测鼠疫耶尔森氏菌的重组LuxAB(‘light’)标记报告噬菌体。目前，疾控中心和世界卫生组织(WHO)将鼠疫杆菌特异性噬菌体作为确诊鼠疫菌鉴定的诊断标准；然而，噬菌体裂解试验是以实验室为基础的，需要2至3天才能完成。相比之下，本提案中描述的技术不需要样品处理、实验室环境或长时间的孵化期。阶段I结果表明，LuxAB报告噬菌体对减毒的鼠疫菌株迅速产生生物发光信号反应，并显示出作为鼠疫诊断的潜力。第二阶段的研究将建立在第一阶段的基础上，目标是制作一套鼠疫诊断试剂盒。第二阶段的研究将：(I)提高报告噬菌体对鼠疫耶尔森菌的特异性和敏感性(目标1)；(Ii)展示报告噬菌体对野生型鼠疫菌株文库的实用性(目标2)；(Iii)使用一组致病的非鼠疫耶尔森氏菌来分析报告噬菌体对鼠疫菌的特异性(目标3)；以及(Iv)分析和优化检测系统直接与临床标本发挥作用的能力(目标4)。总之，这项研究将为鼠疫诊断试剂盒提供基础，该试剂盒使用新型的“发光”报告噬菌体，可以快速、灵敏和特异地赋予鼠疫耶尔森菌生物发光表型。该产品的愿景是，它可以用于诊断疑似鼠疫感染的患者，使用培养的分离物，或直接与实验室环境中感染的临床样本，或作为便携式相机为基础的分析。我们研究的长期目标是建立一种基于报告噬菌体的多重鉴定方法，用于鉴定不同的细菌优先A病原体。 与公共卫生相关：这项提议意义重大，因为它将产生一种快速的鼠疫诊断试剂盒。为了专门诊断疾病、防止疾病传播和挽救生命，快速诊断试剂盒是必不可少的。