Phage-mediated bioluminescent detection of Yersinia pestis

描述（由申请人提供）：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，黑死病是一种传染性疾病，在整个历史过程中造成数百万人死亡。虽然这种疾病的自然发生现在相对罕见，但恐怖组织利用鼠疫杆菌作为生物武器的可能性是真实存在的。由于该病固有的传染性、快速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实施公共卫生措施和启动危机管理至关重要。这项建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。I期研究成功完成了原理验证研究，并产生了一个重组luxAB（“light”）标记的报告噬菌体，用于检测鼠疫杆菌。鼠疫杆菌特异性噬菌体目前被美国疾病控制与预防中心和世界卫生组织（WHO）用作确诊鼠疫杆菌的诊断标准；然而，噬菌体裂解试验是在实验室进行的，需要2到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、实验室环境或广泛的潜伏期。I期试验结果表明，luxAB报告噬菌体对一种减毒鼠疫杆菌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，目标是生产一种鼠疫诊断试剂盒。II期研究将：(i)提高报告噬菌体对鼠疫杆菌的特异性和敏感性（Aim 1）；（ii）证明报告噬菌体对野生型鼠疫杆菌菌株库的效用（目标2）；（iii）使用一组致病性非鼠疫耶尔森菌来分析报告噬菌体对鼠疫杆菌的特异性（目标3），（iv）分析并优化检测系统直接与临床标本一起工作的能力（目标4）。总的来说，这项研究将为使用新型“发光”报告噬菌体的鼠疫诊断试剂盒提供基础，该试剂盒可快速、敏感和特异性地赋予鼠疫杆菌生物发光表型。该产品的愿景是，它可用于诊断疑似鼠疫感染患者的培养分离株，或在实验室环境中直接与受感染的临床标本一起使用，或作为基于便携式相机的测定方法。我们研究的长期目标是开发一种基于报告噬菌体的多重检测方法，用于鉴定不同的细菌优先a病原体。

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.