A PCR Jet for Rapid Detection of Special pathogens

描述（由申请人提供）：DNA的聚合酶链反应（PCR）扩增是快速检测病原体最可靠的方法。PCR过程的总体速率由热循环器和用于酶复制DNA的伴生生物化学的性能决定。在第一阶段的研究中，制作了一个非常快速的实验PCRJet热循环器。这台带电子阀的加压燃气机可以在1分钟内进行30次PCR循环，温度控制+ 0.2°C。热循环器是如此之快，以至于DNA生物化学是限速的。PCRJet已用于诊断炭疽芽孢杆菌和密切相关的蜡样芽孢杆菌，在3.5 ~ 8.5分钟内使用30-40个PCR循环。随着工程技术的改进和实时检测光学，应该可以在< 8分钟内常规扩增和检测特殊病原体。在第二阶段的研究中，将基于计算流体动力学建模制造高性能的PCRJet。该仪器具有高速DNA扩增和荧光染料检测功能，可在5分钟内检测出诊断性炭疽杆菌和天花病毒的主要DNA片段。使用生化工程的方法来优化PCR过程，一个非常快速的热稳定的喷射聚合酶混合物（KOD Pol + dUTPase + PPase）将被配制，因此延伸率>500 nt/秒在72¿C可以实现。结合PCRJet热循环器和改进的酶学技术，用于DNA的高速扩增，将使生物恐怖主义试剂的诊断在<2分钟的时间尺度上。

DESCRIPTION (provided by applicant): 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.