Rapid Turn-arround Multiplex Testing: Bioweapon Agents

描述(由申请人提供)：该项目的目标是开发和验证一个新的生物疫苗检测诊断平台，该平台提供超快的设计和实施。今天，科学界和安全界都认为，生物技术的进步增加了人们对生物武器计划中滥用的担忧。去年年底，随着美国各地炭疽病袭击的报道成倍增加，人们越来越担心可能会出现新的基因组发生变化的菌株。因此，需要新的诊断技术，为以前未知的生物病毒株提供快速周转检测。为此，我们开发了一个新的平台，基因编码2.0，它提供了一个超快的转向实时PCR基因检测的方法。基因编码2.0使用了一个扩展的遗传信息系统(宙斯盾)，它允许在聚合酶链式反应过程中对报告分子进行位点特异性的酶结合。该平台已经向商业市场展示了超灵敏的定量炭疽检测。在第一阶段，我们设计并展示了CDC所有六种A类生物恐怖制剂的平台。所有系统都能够检测到给定生化武器制剂基因组的单一拷贝。 在第二阶段，我们将开发多路系统来分析给定生物疫苗制剂中的多个遗传位点，这将允许制造商以超快的方式改变序列特异性。我们将结合两个试剂特定的检测系统和内部阳性对照。多个特定目标的检测极大地提高了对阳性检测结果的特异性的置信度。内部阳性对照通过确认检测试剂的正确作用，使阴性检测结果可信。这两个特征对于生物武器检测特别重要。一旦多重目标实现，我们将挑战这些系统，用一组令人困惑的DNA样本和环境PCR抑制剂对它们进行测试。我们将展示一种脱水反应配方，它对运输和储存都是稳定的。我们还将开发软件工具，以促进生物制剂的基因密码检测和定量的自动化。当其他目标达到时，我们将在外部地点验证基因密码系统。

DESCRIPTION (provided by applicant): The goal of this project, over Phase I and Phase II, is to develop and validate a new diagnostic platform for biowarfare detection that provides ultrafast design and implementation. Today, both the scientific and security communities believe that advances in biotechnology have increased the concern for misuse in biological weapon programs. As reports of anthrax attacks across the United States multiplied late last year, an increasing concern grew that new strains with altered genomes may appear. Therefore, new diagnostic technologies that provide quick turnaround assays to previously unknown biowarfare strains are needed. To this end, we developed a novel platform, GENE-CODE 2.0, that provides an ultraquick turn-around to real-time PCR genetic testing. GENE-CODE 2.0 employs an expanded genetic information system (AEGIS) that allows for site-specific enzymatic incorporation of reporter molecules during PCR. The platform has already been demonstrated to the commercial market for ultrasensitive quantitative anthrax detection. In Phase I we have designed and demonstrated the platform for all six CDC Category A biological terror agents. All systems were capable of detecting a single copy of a given bioweapons agent genome. In Phase II we will develop multiplexed systems to analyze multiple genetic sites within a given biowarfare agent that will allow the manufacturer to change sequence specificity in an ultrafast manner. We will combine two agent-specific detection systems with an internal positive control. Detection of multiple specific targets greatly improves confidence in the specificity of positive assay results. The internal positive control lends credence to negative assay results by confirming the proper function of assay reagents. Both of these features are especially important for bioweapons detection. Once the multiplex goal is achieved we will challenge the systems by testing them with a panel of confounding DNA samples and environmental PCR inhibitors. We will demonstrate a dehydrated reaction formulation that is stable to shipping and storage. We will also develop software tools to facilitate automation of GENE-CODE detection and quantitation of bioagents. When the other aims are met we will validate the GENE-CODE system at an external site.