Development of a helicase-based rapid DNA diagnostic system for Biodefense

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DESCRIPTION (provided by applicant): Nucleic acid based diagnostic devices allow accurate identification of biological agents by specifically amplifying specific target sequences from environmental samples. Current diagnostic tests rely on PCR, a more costly and time-consuming detection method that cannot be easily adapted for use in the field. An inexpensive, easy-to-use hand-held diagnostic test for the detection of biological agents onsite will allow first responders to rapidly assess a potential bioterrorism threat. Our laboratory has developed an isothermal DNA amplification system that can be performed independently of a thermal cycler. The technology, Helicase-Dependent Amplification (HDA), employs helicase to enzymatically unwind DNA, rather than using high temperature, to expose the target sequence and allow specific primers to anneal, followed by DNA polymerase mediated primer extension. Products from each cycle of HDA enter the next cycle, resulting in exponential amplification. The technology has been demonstrated to detect as little as 100 cells of the bioterrorism agent Bacillus anthracis. With continued research, the sensitivity and efficiency of HDA will be improved upon and used in the next generation of isothermal nucleic acid diagnostic tests. The main objective of the Phase I research is to innovate HDA by determining and addressing the limitations of the HDA platform and to develop a reverse transcription HDA (RT-HDA) system for use in a multiplexed test for the simultaneous detection of DNA and RNA encoded pathogens, such as B. anthracis and Ebola virus. The aims of Phase I will examine devising an efficient primer design protocol, improving HDA by enhancing the performance of HDA enzymes and developing a one-step, isothermal RT-HDA platform. Innovations to the HDA technology in Phase I will allow the development and production of hand-held HDA based diagnostic devices in Phase II. By innovating the quality of diagnostic testing, we seek to provide emergency personnel with the necessary tools to protect communities from potential biological threats.