Polymerase Chain Reaction (PCR) amplification of DNA is the most reliable method for the rapid detection of pathogens. The overall rate of the PCR process is determined by performance of both the thermocycler and the companion biochemistry used to enzymatically copy DNA. In Phase I Research, a very fast experimental PCRJet thermocycler has been fabricated. This pressurized gas machine with electronic valves can carry out 30 PCR cycles with thermal control + 0.2 ?C in <1 minute. The thermocycler is so fast that the DNA biochemistry is rate limiting. The PCRJet has been used to diagnose Bacillus anthracis and closely related B. cereus using 30-40 PCR cycles in 3.5 to 8.5 minutes. With engineering improvements and real-time detection optics, it should be possible to routinely amplify and detect special pathogens in < 8 minutes. In Phase II Research, a high performance PCRJet will be fabricated based on Computational Fluid Dynamic modeling. This machine will be capable of both high-speed DNA amplification and fluorescent dye-based detection, so that diagnostic B. anthracis and Variola major DNA fragments can be detected in ~5 minutes. Using a biochemical engineering approach to optimization of the PCR process, a very fast thermostable Jet Polymerase enzyme mixture (KOD Pol + dUTPase + PPase) will be formulated, so that elongation rates >500 nt/sec at 72?C can be achieved. The combination of the PCRJet thermocycler and improved enzymology for high-speed amplification of DNA will enable diagnosis of agents of bioterrorism on a time-scale of <2 minutes. ? ?
