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.