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的最可靠方法。PCR过程的总体速率由用于酶促复制DNA的热循环仪和伴随生物化学的性能决定。在第一阶段的研究，一个非常快速的实验PCRJet热循环仪已经制造。这种带电子阀的压缩气体机器可以在1分钟内进行30个PCR循环，温度控制在+0.2 ℃。热循环仪是如此之快，以至于DNA生物化学是速率限制。PCRJet已用于诊断炭疽芽孢杆菌和密切相关的B。在3.5至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技术的发展，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，高性能的PCRJet将基于计算流体动力学模型制造。这台机器将能够同时进行高速DNA扩增和基于荧光染料的检测，以便诊断B。炭疽和天花主要DNA片段可以在约5分钟内检测到。使用生物化学工程方法优化PCR过程，将配制非常快速的热稳定Jet聚合酶混合物（KOD Pol + dUTR + PPase），使得可以实现在72 ° C下>500 nt/sec的延伸速率。PCRJet热循环仪和改进的酶学相结合，用于DNA的高速扩增，将能够在<2分钟的时间尺度上诊断生物恐怖主义制剂。

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.