A Rapid, Sensitive and Fully Automated Y. pestis Test

描述(由调查人员提供)：2001年的炭疽热袭击证实了生物恐怖主义是一种真实的可能性。虽然袭击规模不大，但其影响达到了恐怖的程度。不幸的是，正如前参议员沃伦·鲁德曼所指出的，“未来几年在这个国家发生恐怖袭击的可能性很大，而不是不可能”。美国外交关系委员会最近的研究得出结论，我们的国家仍然“没有准备好应对美国领土上的灾难性袭击，特别是涉及化学、生物或核毒剂的袭击”。鼠疫耶尔森氏菌在过去两千年中估计有1亿鼠疫受害者，其病原体出现了对抗生素的耐药性，已被确定为A类生物恐怖主义病原体。 在发生生物恐怖主义袭击时，最重要的第一反应之一是快速识别一个或多个生物剂，并及时诊断那些已感染的人。目前选择的敏感检测鼠疫耶尔森氏菌的方法是聚合酶链式反应(PCR)。然后用培养方法和/或EIA确认该有机体。然而，聚合酶链式反应的使用也并非没有问题。聚合酶链式反应扩增产物可能受到污染，这就需要一个专门的、高度受控的中心实验室，而这又需要长途运送样品。事实上，聚合酶链式反应容易受到抑制性杂质的影响，这需要长时间的样品准备和重复测试。所有这些问题都是在2001年炭疽热恐怖袭击期间遇到的。 我们已经开发出一种快速(大约60分钟)和敏感(不到1个细菌)的败血症检测原型，使用我们的基于微粒的扩增(MBA)技术和一类新的细菌标志物。这一阶段应用的具体目的是研究使用相同技术但不同种类的标记物来开发(A)快速(60-90分钟)、(B)敏感(小于100cfu)、(C)特异性(与其近亲无交叉反应)和(D)全自动的鼠疫耶尔森氏菌检测的可行性。完全开发后，分析的唯一实际步骤是加载临床样本(不一定是纯化的核酸)。由于这些仪器的双重用途，鼠疫耶尔森氏菌检测可以在临床实验室广泛使用，从而在发生生物恐怖主义事件时提供第一线诊断测试。

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.