Multiplex PCR Detection of CDC 'A' Bioterrorism Agents

描述（由申请人提供）：炭疽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构成了日益可怕的威胁。疾病预防控制中心（“A”类）确定的构成最大威胁的病原体包括大天花（天花）、炭疽芽孢杆菌（炭疽）、鼠疫耶尔森氏菌（鼠疫）、肉毒杆菌毒素（肉毒杆菌中毒）、土拉菌弗朗西斯菌（土拉菌病）和一组引起出血热的RNA病毒（vhf，例如埃博拉病毒）。准确和有效地识别和诊断这些药物的技术严重有限。缺乏良好的诊断测试阻碍了NIAID和CDC为美国应对未来的生物恐怖袭击而制定的大部分目标。现有的旧技术已被证明不可靠。现代分子测试，如单个PCR分析，已经发展为一些试剂。这些技术提高了检测速度和灵敏度，但由于有太多的生物恐怖主义制剂，在每个样本上运行数十个“单路”阵列是令人望而却步的。同样，最近报道的微芯片（MAGI Chip）阵列和其他微阵列要么需要先进行PCR扩增，要么成本高昂，而且需要复杂的设备。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制剂的检测方法，将极大地加强反恐计划和生物防御。我们的实验室开创了一种多重PCR方法，可以实现这一目标。这种专有方法（两项美国专利）已在Hexaplex(r) Assay中商业化使用，该方法可以在一次测试中检测七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的独特技术进行多重pcr -酶杂交测定（EHA），该技术将识别所有CDC“a”类生物恐怖主义制剂的DNA；2) RNA基；最后3)一个单一的组合多重（RNA/DNA） PCR检测，其分析灵敏度等同于CDC开发的“单一”实时检测。具体目标4：确定在临床试验中这种多重检测是否等同于这些“单一”检测。

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.