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类优先细菌性病原体。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。该应用程序的长期目标和商业应用是开发一种鼠疫诊断检测试剂盒。这项I期研究的目的是产生一种luxAB（“光”）标记的噬菌体，可以特异性检测鼠疫杆菌。鼠疫杆菌特异性噬菌体目前被疾病预防控制中心和世界卫生组织（世卫组织）用作确认鼠疫杆菌鉴定的诊断标准；然而，噬菌体裂解试验是基于实验室的，需要18-24小时才能完成。相反，本应用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。Specific Aim 1将通过将细菌luxAB基因整合到鼠疫诊断噬菌体基因组的非编码区，产生鼠疫杆菌报告噬菌体。重组luxab标记的噬菌体将根据感染培养物发出生物发光的能力进行鉴定和分离。Specific Aim 2将进行可行性研究，以证明luxab -噬菌体可以有效地用作鼠疫杆菌检测系统。将使用减毒鼠疫杆菌菌株（豁免选择剂，BSL2病原体）。将确定检测的灵敏度极限、信号响应时间和剂量-响应特性。总的来说，这一申请将为一种新型“产光”报告噬菌体的主要研究提供证据，这种噬菌体可以快速和特异性地检测鼠疫杆菌。我们的初步结果已经证明了一种类似的基于方法检测非封装的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年NIAID生物防御研究战略计划），Guild Associates的长期目标之一是开发一种报告噬菌体鸡尾酒，可用于诊断鉴定不同的a类细菌病原体，如炭疽芽胞杆菌和鼠疫杆菌。公共卫生相关性：这一应用具有重要意义，因为它将为鼠疫快速诊断检测试剂盒产生原理证明结果。快速诊断检测试剂盒对于迅速实施可挽救生命和防止疾病传播的公共卫生措施至关重要。

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.