Bioagent Characterization by Iterative Extension

DESCRIPTION (provided by applicant): Project Summary - A new syndrome-based diagnostic system for rapid detection and identification of category B and C priority pathogens is proposed. Phase 1 will focus on V. cholerae genes and genes from similar pathogens. The technique relies upon a helicase-driven iterative primer extension reaction (HDIE) that uses isothermal, multiplexed target amplification to produce short, labeled, single-stranded DNA products with sequences representative of the sample DNA. These reaction products are ideal for hybridization to short probes immobilized on microarrays, thus providing gene identification of any pathogen in the sample. Phase II will expand the technique to a wide variety of category B and C priority pathogens. Successful completion of the research will result in a syndrome-based diagnostic system that can identify one or more pathogens in a sample among a large number of candidates. The technique will be isothermal and require minimum equipment for the initial reactions, making it useful to first responders in a biowarfare attack or an outbreak. HDIE relies on the fact that there are a large number of regions in any gene that are missing one of the four DNA bases over a span of 12 to 40 bases. HDIE primers are designed to be complementary to a section of the target DNA immediately adjacent to the 3' end of one of these void regions. Once the primer is annealed to the target DNA, the extension reaction proceeds from the 3' end of the primer using the region lacking either A, T, G, or C as the template. When the first occurrence of the missing base is encountered the extension reaction is terminated by incorporation of a complementing dideoxynucleotide. DNA helicases separate the extension reaction products from the template molecules, allowing iterative non-exponential target amplification. The HDIE products can be hybridized to microarray probes and detected using conventional fluorescence methods. The fact that the reaction is terminated after a very short extension, along with fact that amplification is linear rather than exponential, allows hundreds of primers to be present in a single reaction tube. Although the HDIE approach is designed to overcome difficulties specific to parallel pathogen diagnosis, it can be extended to many applications that tap into the lucrative molecular diagnostics market, including foodborne pathogen detection, agricultural and veterinary testing, water testing, and clinical testing. In 2005, the global market for molecular diagnostics is expected to be $6.5 billion, or about 3.3% of the total diagnostics market and approximately 14% of the in vitro diagnostic market. The molecular diagnostics market is forecast to increase to $12 billion by 2010 and $35 billion by 2015. Relevance - The proposed research directly addresses the need for a syndrome-based assay that can rapidly identify' the pathogen or pathogens responsible for disease in case of a biowarfare attack or a major outbreak.