A Rapid, Sensitive and Fully Automated Y. pestis Test

描述（由调查人员提供）：2001年炭疽袭击证实，生物恐怖主义是一个真实的可能性。这次袭击虽然规模很小，但其影响却达到了恐怖的程度。不幸的是，正如前参议员沃伦·鲁德曼所指出的那样，“未来几年我国发生恐怖袭击的可能性大于不可能性”。 外交关系理事会最近的一项研究得出结论，我国仍然“没有准备好应对对美国领土的灾难性攻击，特别是涉及化学、生物或核制剂的攻击”。在过去的两千年里，估计有1亿鼠疫受害者，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类生物恐怖主义制剂。 在发生生物恐怖主义袭击时，最重要的第一反应之一是迅速查明生物制剂，并及时诊断受感染者。目前用于鼠疫耶尔森氏菌敏感检测的方法是那些涉及聚合酶链反应（PCR）的方法。然后用培养方法和/或EIA确认微生物。然而，PCR的使用并非没有问题。PCR扩增子的可能污染需要一个专门的和高度控制的中心实验室，这反过来又需要长距离的样本运输。PCR易受抑制性杂质的影响，这一事实需要长时间的样品制备和重复检测。所有这些问题在2001年炭疽恐怖袭击期间都遇到过。 我们已经开发了一种快速（约60分钟）和敏感（少于1个细菌）的原型败血症测试使用我们的微粒为基础的扩增（MBA）技术和一类新的细菌标志物。第一阶段申请的具体目的是研究使用相同技术但不同种类的标记物开发鼠疫耶尔森氏菌检测的可行性，该检测是（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），和（d）全自动的。当完全开发时，检测的唯一动手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，鼠疫耶尔森氏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。

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.