The goal of this research application is to develop a safe, easy to use, high-throughput, automated nucleic acid testing system based on our proprietary """"""""Lab-in-a-tube"""""""" (Liat TM) Technology. The Liat system enables the integration of sample preparation, multiplex DNA amplification and real-time quantification in a single closed-tube system. Furthermore, the technology enables very rapid and efficient thermal cycling on large sample volumes. The enrichment of target DNA from whole blood and other fluids using a closed-tube processing method has also been demonstrated. A compact, closed-system design ensures the system's ability to be rapidly deployed in mobile or forward-testing labs for the detection of bio-warfare agents, and for rapid diagnosis during an infectious disease outbreak. The proposed system will also provide widespread benefits to clinical sample testing for sexually transmitted diseases, viral load testing, and therapeutic monitoring. Furthermore, the speed, throughput capacity, and thermal flexibility of the system make it an ideal tool for rapid assay development. As an extension of our handheld Molecular Analyzer, the proposed Liat Molecular Workstation will be developed and constructed for the simultaneous processing of 48 samples. Phase-I research will focus on' the integration of rapid, real-time, closed-tube amplification. A tube strip containing pre-packed reagent and efficient optical coupling means will be developed and tested. Following an initial performance validation, the system will be characterized for speed, sensitivity, specificity, dynamic range, and multiplexing capability. This will be performed using detection probes to score a novel SNP that has been shown to differentiate between B. anthracis and B. thuringiensis. The performance results will then be compared to those obtained using two commercial systems. We believe this proposed research will contribute significantly to two of NIAID's top priorities: 1) improving our nation's ability to respond to biological warfare threats and, 2) enhancing the diagnosis and treatment of infectious diseases.
