A Rapid, Sensitive and Fully Automated Y. pestis Test

快速、灵敏且全自动的鼠疫耶尔森氏菌测试

• 批准号: 6887444
• 负责人: XING-XIANG LI
• 金额: $ 19.44万
• 依托单位: CELLEX, INC.
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-05-01 至 2006-10-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6887444
• 关键词: Yersinia pestis; Yersinia pestis disease; bacterial RNA; bacterial genetics; biomarker; biomedical automation; biomedical equipment development; bioterrorism /chemical warfare; clinical biomedical equipment; clinical research; diagnosis design /evaluation; diagnostic tests; early diagnosis; human tissue; nucleic acid probes; nucleic acid sequence; polymerase chain reaction; tissue /cell culture; transfer RNA

DESCRIPTION (provided by investigator): The 2001 Anthrax attack confirmed that bioterrorism is a real possibility. Although the attack occurred at a small scale, its impact reached terror proportion. Unfortunately, as former Senator Warren Rudman pointed out, "the likelihood of a terrorist attack in this country in the next several years is more likely than unlikely". Recent study by the Council on Foreign Relations concluded that our nation remains "unprepared to handle a catastrophic attack on American soil, particularly one involving chemical, biological or nuclear agents". With an estimated 100 million plague victims in the last two millennia and the emergence of antibiotic resistance of its causative agent, Yersinia pestis has been identified as a category A bioterrorism agent. In the event of a bioterrorism attack, one of the most important first responses is rapid identification of the biological agent or agents, and timely diagnosis of those who have been infected. Current methods of choice for sensitive detection of Yersinia pestis are those involving polymerase chain reaction (PCR). The organism is then confirmed with culture methods and/or EIA. The use of PCR is not without problems, however. Possible contamination of PCR amplicons necessitates a specialized and highly controlled central laboratory, which in turn necessitates long distance sample shipping. The fact that PCR is susceptible to inhibitory impurities requires lengthy sample preparation and duplicate testing. All these problems had been encountered during the 2001 Anthrax terror attack. We have developed a rapid (approximately 60 minutes) and sensitive (less than 1 bacterium) prototype sepsis test using our microparticle based amplification (MBA) technology and a novel class of bacterial markers. The specific aim for this Phase I application is to study the feasibility of using the same technology but different species of markers to develop a Yersinia pestis test that is (a) rapid (60-90 minutes), (b) sensitive (less than 100 CFU), (c) specific (no cross reactivity with its close relatives), and (d) fully automatic. When fully developed, the only hands-on step for the assay is the loading of clinical samples (not necessarily purified nucleic acids). Because of the dual use of the instruments, the Yersinia pestis test could be widely available in clinical laboratories and thus offer a first-line diagnosis testing in the event of a bioterrorism.

描述（由调查员提供）：2001 年炭疽袭击证实生物恐怖主义确实存在可能。这次袭击虽然规模不大，但影响却达到了恐怖程度。不幸的是，正如前参议员沃伦·鲁德曼指出的那样，“未来几年这个国家发生恐怖袭击的可能性大于可能性”。 美国外交关系委员会最近的研究得出的结论是，我们的国家仍然“没有准备好应对对美国领土的灾难性袭击，特别是涉及化学、生物或核制剂的袭击”。过去两千年里估计有 1 亿鼠疫受害者，并且其病原体出现了抗生素耐药性，鼠疫耶尔森氏菌已被确定为 A 类生物恐怖主义病原体。 在发生生物恐怖袭击时，最重要的第一反应之一是快速识别生物制剂，并及时诊断感染者。目前选择的灵敏检测鼠疫耶尔森氏菌的方法是涉及聚合酶链式反应 (PCR) 的方法。然后通过培养方法和/或 EIA 确认生物体。然而，PCR 的使用并非没有问题。 PCR 扩增子可能受到污染，因此需要专门且高度控制的中心实验室，而这又需要长距离样本运输。 PCR 容易受到抑制性杂质的影响，因此需要漫长的样品制备和重复测试。所有这些问题都在2001年炭疽恐怖袭击中遇到过。 我们使用基于微粒的扩增 (MBA) 技术和一类新型细菌标记物开发了一种快速（约 60 分钟）且灵敏（少于 1 个细菌）的败血症原型测试。第一阶段应用的具体目的是研究使用相同技术但不同物种标记来开发鼠疫耶尔森氏菌测试的可行性，该测试具有 (a) 快速（60-90 分钟）、(b) 灵敏（小于 100 CFU），（c）特异性（与其近亲无交叉反应性），以及（d）全自动。完全开发后，检测的唯一实际操作步骤是加载临床样品（不一定是纯化的核酸）。由于仪器的双重用途，鼠疫耶尔森菌测试可以在临床实验室广泛使用，从而在发生生物恐怖主义时提供一线诊断测试。