Bacillus anthracis was used by bio-terrorists to attack the civil and governmental institutions three years ago. B. anthracis is indeed a potential biological warfare agent because it is highly pathogenic and easy to cultivate and produce, and because its spore form is highly resistant to inactivation. B. anthracis inhalation and gastrointestinal infection most likely lead to death if the infected patients are not treated promptly. To ensure immediate medical treatment, rapid and accurate detection of B. anthracis in field and clinical settings is essential. To meet the need, we have developed a direct detection system for specific RNAs in a total RNA sample via RNA-DNA hybrid template binding, RNase H digestion, and Klenow 3'- extension/labeling. The detection sensitivity can reach up to attomole (10[-18 ]mole) level. This novel approach of RNA detection in microplate format doesn't require reverse transcription, polymerase chain reaction, laser excitation, fluorescence detection, transcription, and/or gel electrophoresis. To develop a sensitive and specific pathogen detection strategy, I propose here to develop an RNA microchip methodology and technology on the base of this RNA 3'-labeling/detection system, by immobilizing the RNA-DNA hybrid temples that recognize B. anthracis signature RNAs on a microchip. In brief, the signature RNAs are hybridized to this microchip while non-specific RNAs are washed away. The bound RNAs are labeled with biotin on this chip via RNase H digestion and Klenow labeling. The biotin labels are then converted to the enzyme labels with the antibiotin antibody-alkaline phosphatase (AP) conjugate. Finally, the labeled RNAs are detected by the AP-catalyzed chemiluminescent reaction and chemiluminescence detection. Bacillus anthracis can thus be positively identified via signature RNA detection on this RNA microchip. After optimization of the detection system, the pathogen detection can be achieved in twenty minutes or shorter time. This simple and rapid RNA microchip technology will also be used in the future for multiple pathogen and disease detection in biodefense and in clinical settings, for point-of-care diagnosis, for rapid SNP analysis and individualized medicine administration, for cancer detection and subtype classification, and for food, air and water safety monitoring. These long-term goals are beyond the scope of this proposal.
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