Rapid Turn-arround Multiplex Testing: Bioweapon Agents

描述（由申请人提供）：该项目的目标，在第一阶段和第二阶段，是开发和验证一个新的诊断平台，生物战检测，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对生物武器计划中滥用的关注。随着去年年底美国各地炭疽袭击的报道成倍增加，人们越来越担心可能会出现基因组改变的新菌株。因此，需要新的诊断技术，为以前未知的生物战菌株提供快速周转测定。为此，我们开发了一个新的平台GENE-CODE 2.0，为实时PCR基因检测提供了一个超快速的周转。GENE-CODE 2.0采用了扩展的遗传信息系统（AEGIS），允许在PCR过程中位点特异性酶促掺入报告分子。该平台已经在商业市场上进行了超灵敏定量炭疽检测的演示。在第一阶段，我们设计并演示了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物武器试剂基因组的单个拷贝。 在第二阶段，我们将开发多路复用系统来分析给定生物战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。我们将联合收割机两种试剂特异性检测系统与内部阳性对照组合。多个特异性靶标的检测大大提高了阳性检测结果特异性的置信度。内部阳性对照通过确认试验试剂的正确功能，为阴性试验结果提供依据。这两个特征对于生物武器检测尤其重要。一旦实现了多重目标，我们将通过用一组混淆的DNA样本和环境PCR抑制剂测试系统来挑战它们。我们将证明一种脱水的反应配方，它对运输和储存是稳定的。我们还将开发软件工具，以促进基因编码检测和生物制剂定量的自动化。当其他目标得到满足时，我们将在外部站点验证GENE-CODE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.