Recent worldwide events have highlighted the need for diagnostic tools to identify bioterrorism agents and pathogens causing human diseases. Current detection methods for those agents do not satisfy the need for high sensitivity, specificity and speed in the detection of those agents. This application proposes to develop and validate a microarray based rapid pathogen detection method (less than 4 hours) that can be used to type multiple pathogens in a single experiment, with high sensitivity and specificity. In addition, this technology will be used to identify genetically manipulated strains, through the detection of antibiotic resistance and toxin genes. Affymetrix and the Institut Pasteur in France propose to collaborate on the development of this application. The goal of the project is to be able to identify multiple pathogens (bacteria and viruses) simultaneously, but also to identify genetic manipulations in these organisms. Early detection of biological agents and genetic manipulations will allow for earlier initiation of defense mechanisms, evacuations and a fast medical response for the affected areas and people. Specifically, we propose to: 1. Design a high-density oligonucleotide resequencing array for the identification of 18 bacterial pathogens from the NIAID priority list using their unique 16S rRNA gene sequence, and 25 viruses using organism specific gene sequences; 2. Design a high-density oligonucleotide expression array for the identification of genetically manipulated organisms using known toxin and antibiotic resistant genes; 3. Develop and test a target preparation method that utilizes universal PCR amplification for the selected bacterial pathogens, a multiplex amplification system for viral organisms and a whole genome amplification method to detect genetic elements like toxins and antibiotic resistance genes; 4. Develop a sample preparation and hybridization protocol which, combined, takes less than 4 hours to complete; and 5. Develop and test novel algorithms for the analysis of the pathogen resequencing array to automatically identify and type individual bacteria and viruses in clinical samples and to detect potential genetic elements in these organisms.
