Bioluminescent reporter phage for the diagnostic detection of shigellosis

用于志贺氏菌病诊断检测的生物发光报告噬菌体

• 批准号: 8839507
• 负责人: DAVID A SCHOFIELD
• 金额: $ 42.96万
• 依托单位: GUILD ASSOCIATES, INC.
• 依托单位国家: 美国
• 财政年份: 2014
• 资助国家: 美国
• 起止时间: 2014-06-01 至 2017-05-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/8839507
• 关键词: Accounting; Acute; Antibiotic susceptibility; Area; Bacteria; Bacteriophages; Binding; Biochemical; Biological Assay; Bioterrorism; Categories; Cause of Death; Cells; Centers for Disease Control and Prevention (U.S.); Cessation of life; Characteristics; Child; Chromosomes; Clinical; Collection; Communicable Diseases; DNA; Data; Detection; Development; Devices; Diagnosis; Diagnostic; Disease; Drug resistance; Dysentery; Environment; Enzymes; Epidemic; Freeze Drying; Generations; Genes; Genetic Engineering; Genome; Goals; Grant; Growth; Hand; Health; Human; Infection; Lead; Light; Luciferases; Mediating; Methods; Morbidity - disease rate; Multi-Drug Resistance; Operating System; Patients; Phage Display; Phase; Phenotype; Process; Receptor Cell; Recombinants; Reporter; Reporter Genes; Research; Resources; Sampling; Serological; Shigella; Shigella Infections; Shigella boydii; Shigella dysenteriae; Shigella flexneri; Shigella sonnei; Signal Transduction; Site; Species Specificity; Specificity; Specimen; Staging; System; Technical Expertise; Technology; Testing; Translating; antimicrobial drug; biodefense; clinical Diagnosis; cost; enteric pathogen; fighting; improved; mortality; outcome forecast; pandemic disease; rapid detection; response; sample collection; tool; transmission process; trend

Shigellosis is a global human health problem and a biodefense area of concern. The disease, caused by Shigella species, is a significant cause of morbidity and mortality accounting for 164 million cases worldwide and 1.1 million deaths annually, most notably amongst children. The disease is extremely infectious, requiring only 10-100 bacterial cells to initiate infection. Moreover, there is a global rise in the occurrence of multi-drug resistant isolates, such as the epidemic and pandemic Shigella dysenteriae type 1 strain. Despite this trend, the standard methods for the confirmed identification of the disease and the determination of antibiotic susceptibility, are traditional culturing methods, followed by serological and biochemical tests; these assays require 24-48 h to complete in a lab environment. The long-term goal of this research is to develop a rapid, and portable diagnostic assay technology that can detect the causative agents of shigellosis. Importantly, the diagnostic technology will be able to simultaneously provide an antibiotic susceptibility profile, which will enable appropriate treatment options and thus lead to improved patient prognosis. The R21 phase will generate the proof-of-principle results for the development of ¿light-tagged¿ Shigella reporter phages that can detect Shigella spp. by specifically conferring a bioluminescent signal response. Aim 1 will identify and prioritize Shigella phages which display species specificity and broad strain infectivity. Aim 2 will integrate the bacterial luxAB reporter genes into non-essential sites of the prioritized Shigella phage genomes to create luxAB-tagged reporter phages. In the presence of target bacteria, the reporter phage bind to specific cell receptors, inject their phage DNA, and use the host¿s transcriptional and translational machinery to produce the luciferase enzyme. Upon substrate addition, the ensuing bioluminescent response can be readily detected. Following the demonstration that the Shigella reporter phages have the necessary detection attributes, the R33 grant will develop the reporter phage technology into a clinical diagnostic for shigellosis. We hypothesize that the ¿bioluminescent¿ phage detection system will be able to: (i) rapidly (within minutes) detect the presence of Shigella and differentiate between the species; (ii) require significantly fewer cells to achieve a positive signal (more sensitive); (iii) function directly with clinical specimens (does not require the isolation of pure bacterial cultures); (iv) provide concurrent antibiotic susceptibility data (and help patient prognosis), and (v) function with a simple handheld detection device (field appropriate/battery operated system). The cost of producing the reporter phage and the consumable costs are minimal. The assay does not require technical expertise or processing. Consequently, we believe the technology is particularly well suited to resource-limited settings and will be able to function in a non-laboratory environment as per the requirements set forth in this RFA.

志贺氏菌病是一个全球性的人类健康问题，也是一个值得关注的生物防御领域。这种疾病是由 志贺氏菌属是导致世界范围内1.64亿病例发病和死亡的重要原因 每年有110万人死亡，尤其是儿童。该疾病具有极强的传染性，需要 只有10-100个细菌细胞开始感染。此外，多药耐药的发生率在全球范围内有所上升。 耐药分离株，如流行性和大流行性志贺氏菌1型菌株。尽管有这种趋势， 确定疾病鉴定和抗生素测定的标准方法 敏感性，是传统的培养方法，其次是血清学和生化试验;这些测定 在实验室环境中需要24-48小时完成。 这项研究的长期目标是开发一种快速、便携的诊断分析技术， 检测志贺氏菌病的病原体。重要的是，诊断技术将能够同时 提供抗生素敏感性谱，这将使适当的治疗选择，从而导致 改善患者预后。R21阶段将为以下开发产生原理验证结果： 可以检测志贺氏菌属的光标记志贺氏菌报告菌株。通过特别赋予生物发光 信号响应。目的1将鉴定和优先考虑志贺氏菌，其显示种特异性和广泛的 菌株传染性。目的2将细菌luxAB报告基因整合到优先的非必需位点， 志贺氏菌噬菌体基因组，以创建luxAB标记的报告基因。在靶细菌存在的情况下， 报告噬菌体结合到特定的细胞受体，注入它们的噬菌体DNA，并利用宿主的转录和 翻译机器以产生荧光素酶。加入底物后，随后的生物发光 可以很容易地检测到响应。继志贺氏菌报告者证明， 必要的检测属性，R33赠款将开发报告噬菌体技术进入临床 志贺氏菌病的诊断 我们假设生物发光噬菌体检测系统将能够：（i）快速（在几分钟内） 检测志贺氏菌的存在并在物种之间进行区分;（ii）需要显著较少的细胞， 实现阳性信号（更灵敏）;（iii）直接使用临床样本（不需要 分离纯细菌培养物）;（iv）提供同期抗生素敏感性数据（并帮助患者 预后），以及（v）具有简单手持检测设备的功能（现场适用/电池供电 系统）。生产报告噬菌体的成本和消耗成本是最小的。该测定不 需要技术专长或加工。因此，我们认为这项技术特别适合于 资源有限的设置，并将能够在非实验室环境中运行的要求 在这个RFA中，