Expensive equipment, highly-trained personnel, and the need for a clinical laboratory setting precludes routine nucleic acid testing (NAT) for infectious disease in most of the developing world and even in many resource-limited parts of the United States, leading to wide disparities in health care worldwide. A fast, sensitive, low cos but facile NAT method for robust detection of specific agents at point of care (POC) would help bring molecular diagnostics to everyone. The goal of this application is to demonstrate feasibility of a complete field-appropriate NAT diagnostics system for detection of viral hemorrhagic fever (VHF) RNA for low resource settings. The innovative technology that is the basis of this application is a new thermostable polymerase with innate reverse transcriptase (RT) activity called PyroScript. PyroScript can perform a promising NAT alternative to the polymerase chain reaction (PCR) called loop mediated isothermal amplification (LAMP). Since LAMP is isothermal it does not require specialized instrumentation plus it is much faster than PCR. LAMP is also resistant to inhibitors in crude sample preparations. The PyroScript polymerase is the only known thermostable enzyme combining both strand displacement activity for LAMP-based amplification and RT activity to amplify directly from RNA. The technical advantages of this enzyme are: 1) A single stable enzyme unlike methods requiring two or more labile enzymes to detect RNA. 2) Thermophilic PyroScript can be denatured at 95C. 3) Rapid isothermal amplification from RNA with PyroScript in under 30 minutes. The goal of the proposed research is to further improve PyroScript NAT reagent formulations to provide field-capable stability performance. Another goal is to develop a nucleic acid sample preparation method with ease of use suitable for point of care implementation. These innovations will be combined into locally relevant diagnostic panels that can be performed to test for pathogens endemic to a specific region. By combining Lucigen's capacity in amplification and detection with the expertise in VHF research and fundamental capability provided by the Galveston National Laboratory a NAT system will be developed for detection of viral RNA that can be implemented almost anywhere, worldwide. PUBLIC HEALTH RELEVANCE: A fast, sensitive, low cost but facile nucleic acid test (NAT) method for robust detection of specific agents at point of care would bring the power of molecular diagnostics to low resource settings.