Multiplex PCR Detection of CDC 'A' Bioterrorism Agents

描述(申请人提供)：炭疽病和其他生物战制剂最近受到了强烈的宣传。这些武器对我们的文明来说是一个越来越可怕的危险。疾控中心确定的构成最大威胁的病原体(A类)包括天花(天花)、炭疽芽孢杆菌(炭疽)、鼠疫杆菌(鼠疫)、肉毒杆菌毒素(肉毒杆菌中毒)、图拉氏菌(图拉氏菌)和一组引起出血热的核糖核酸病毒(如埃博拉病毒)。识别和诊断这些病原体的准确和有效的技术严重受限。缺乏良好的诊断测试阻碍了NIAID和CDC制定的大多数目标，这些目标是让美国准备好应对未来的生物恐怖主义袭击。事实证明，可用的旧技术不可靠。现代分子检测，如单独的聚合酶链式反应分析，已经为一些试剂开发出来。这些方法提供了更高的速度和灵敏度，但由于有如此多的生物恐怖主义试剂，在每个样本上运行数十个“单一复合体”阵列是不可能的。同样，最近报道的微芯片(MAGI Chip)阵列和其他微阵列要么需要首先进行PCR扩增，要么需要高成本制造阵列，以及需要复杂的设备。一种(或两种)能够快速、灵敏、特异、廉价地检测大量生物恐怖主义毒剂的方法将极大地加强反恐规划和生物防御。我们实验室开创了一种可以实现这一目标的多重聚合酶链式反应方法。这种专利方法(两项美国专利)已被用于Hexaplex(R)检测，它可以在一次测试中检测出7种常见的呼吸道病毒。该项目的具体目标是：1)确定是否可以使用我们独特的技术进行多重PCR-酶杂交分析(EHA)，该技术将识别所有基于DNA的CDC“A”类生物恐怖分子；2)基于RNA；以及最后3)单一组合多重(RNA/DNA)PCR分析，其分析灵敏度与CDC开发的“Singleplex”实时分析相当。具体目标4：在临床试验中确定这种多重分析是否等同于这些“单链”分析。

DESCRIPTION (provided by applicant): Anthrax and other agents of biological warfare have recently received intense publicity. These weapons are an increasingly fearsome danger to our civilization. Agents identified by the CDC (category "A") to pose the greatest threat include Variola major (smallpox), Bacillus anthracis (anthrax), Yersinia pestis (plague), Clostridium botulinum toxin (botulism), Francisella tularensis (tularemia), and a group of RNA viruses that cause hemorrhagic fevers (VHFs, e.g., Ebola). Accurate and efficient techniques to identify and diagnose these agents are severely limited. This lack of good diagnostic tests hampers the majority of goals set forth by the NIAID and CDC to prepare the U.S. to counter future bioterrorism attacks. Available older techniques have proven unreliable. Modern molecular tests like individual PCR assays have been developed for some agents. These offer increased speed and sensitivity but because there are so many bioterrorism agents it is prohibitive to run dozens of "singleplex" arrays on each specimen. Similarly, recently reported microchip (MAGI Chip) arrays and other microarrays suffer from either needing PCR amplification first, or from the high cost to make the arrays, and the need for sophisticated equipment. A single assay (or two) that could detect a large number of bioterrorism agents rapidly, sensitively, specifically, and cheaply would greatly enhance antiterrorism planning and biodefense. Our laboratory has pioneered a method of multiplex PCR that can accomplish this goal. This proprietary method (two U.S. patents) has been used commercially in the Hexaplex(r) Assay, which can detect seven common respiratory viruses in a single test. The Specific Aims of this project are: 1) To determine if a multiplex PCR-enzyme hybridization assay (EHA) can be made using our unique technology that will identify all of the CDC Category "A" Bioterrorism agents that are DNA based; 2) RNA based; and finally 3) a single combined multiplex (RNA/DNA) PCR assay with an analytical sensitivity equal to "singleplex" real time assays as developed by the CDC. Specific Aim 4: To determine if this multiplex assay is equivalent to these "singleplex" assays in a clinical trial.