Multiplex PCR Detection of CDC 'A' Bioterrorism Agents

CDC“A”生物恐怖主义制剂的多重 PCR 检测

• 批准号: 6597190
• 负责人: Kelly J. Henrickson
• 金额: $ 45.81万
• 依托单位: MEDICAL COLLEGE OF WISCONSIN
• 依托单位国家: 美国
• 财政年份: 2003
• 资助国家: 美国
• 起止时间: 2003-04-01 至 2006-03-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6597190
• 关键词: Bacillus anthracis; Ebola virus; Francisella tularensis; Hantavirus; Lassa virus; Marburg virus; Rift Valley fever virus; Yersinia pestis; bacterial DNA; bacterial disease; biohazard detection; bioterrorism /chemical warfare; communicable disease diagnosis; cooperative study; dengue virus; diagnosis design /evaluation; human subject; patient oriented research; polymerase chain reaction; smallpox virus; varicella zoster virus; virus RNA; virus diseases; yellow fever virus

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.

描述（由申请人提供）：炭疽和其他生物战制剂最近受到了广泛的关注。这些武器对我们的文明构成越来越可怕的威胁。 CDC（“A”类）确定构成最大威胁的病原体包括大天花（天花）、炭疽杆菌（炭疽）、鼠疫耶尔森氏菌（鼠疫）、肉毒杆菌毒素（肉毒杆菌中毒）、土拉弗朗西斯菌（兔热病）和一组引起出血热的 RNA 病毒（VHF，例如埃博拉病毒）。识别和诊断这些病原体的准确有效的技术受到严重限制。缺乏良好的诊断测试阻碍了 NIAID 和 CDC 为美国应对未来生物恐怖主义袭击而制定的大部分目标。现有的旧技术已被证明是不可靠的。现代分子测试，如单独的 PCR 检测，已经针对某些药物进行了开发。这些提供了更高的速度和灵敏度，但由于存在如此多的生物恐怖分子，因此在每个样本上运行数十个“单重”阵列是禁止的。类似地，最近报道的微芯片（MAGI芯片）阵列和其他微阵列要么首先需要PCR扩增，要么由于制造阵列的高成本以及需要复杂的设备而受到困扰。一次（或两次）检测可以快速、灵敏、特异且廉价地检测大量生物恐怖主义制剂，这将极大地增强反恐规划和生物防御。我们的实验室开创了一种可以实现这一目标的多重 PCR 方法。这种专有方法（两项美国专利）已在 Hexaplex(r) Assay 中得到商业应用，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独特的技术进行多重 PCR 酶杂交测定 (EHA)，该技术将识别所有基于 DNA 的 CDC 类别“A”生物恐怖主义制剂； 2) 基于RNA；最后 3) 单一组合多重 (RNA/DNA) PCR 测定，其分析灵敏度等于 CDC 开发的“单重”实时测定。具体目标 4：确定该多重检测是否等同于临床试验中的这些“单重”检测。