Multiplexed Detection of Bioterror Agents

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DESCRIPTION (provided by applicant): The current biothreat to our nation requires the ability to rapidly detect and distinguish bioweapon agents from normal pathogens. Existing detection systems have a limited ability to simultaneously screen in a single sample for multiple agents and their antibiotic resistance, toxin, or virulence genes. To meet this need we propose to use ligase detection reaction (LDR) techniques combined with PCR, capillary electrophoresis, and Universal Arrays, which we have already validated in the detection of cancer gene mutations and the diagnosis of genetic diseases. The studies will include the evaluation of Quantum-dots (Q-dots) as a novel detection approach. The above techniques will be used to meet the specific aims of this proposal: Aim 1: To identify multiple blood-borne pathogens simultaneously, in a single assay. Samples will be evaluated for 16 common bacterial pathogens as well as four blood-borne bioterror agents (B. anthracis, Y. pestis, F. tularensis, B. abortus). Blood specimens from patients with suspected bacteremia will be evaluated in the microbiology laboratory and tested using the above molecular techniques. Random samples will also be spiked with DNA of bioterror pathogens to simulate infection with these agents. Likewise, HIV RNA will be used as a surrogate for RNA hemorrhagic fever viruses. Aim 2: To evaluate antibiotic resistance, enhanced virulence, or genetic manipulation in a positive blood culture. LDR/PCR will be used to detect the presence of the vanA, vanB, mecA, tetL, tetM, gyrA and grlA mutations, additional antibiotic resistance determinants, virulence and toxin genes, which may be present in the common blood-borne pathogens identified in Aim 1. Aim 3: To distinguish Category ABC, engineered, or emergent biothreat agents from common pathogens employing high throughput screening platforms using microbial signature profiles. Standardized protocols using common liquid handling robotic platforms will be established using the techniques validated in Aims 1 and 2. The test will be validated for detecting the above pathogens and further viral agents: Ebola, Marburg, Lassa, Crimean-Congo, Hemorrhagic Fever, Rift Valley Fever, Yellow Fever, SARS Coronavirus, Dengue, and West Nile virus, as well as to distinguish Variola from Vaccinia. In addition, the LDR/PCR virulence gene test from Aim 2 will be expanded to include the major BT toxin and virulence genes. Once verified, our tests will be validated with clinical samples at the CDC.