A PCR Jet for Rapid Detection of Special pathogens

用于快速检测特殊病原体的 PCR Jet

• 批准号: 6832593
• 负责人: NISHA V PADHYE
• 金额: $ 39.49万
• 依托单位: MEGABASE RESEARCH PRODUCTS
• 依托单位国家: 美国
• 财政年份: 2004
• 资助国家: 美国
• 起止时间: 2004-09-15 至 2006-08-31
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6832593
• 关键词: Bacillus anthracis; DNA directed DNA polymerase; biochemistry; biomedical equipment development; biotechnology; bioterrorism /chemical warfare; chemical kinetics; clinical biomedical equipment; communicable disease diagnosis; communicable diseases; high throughput technology; polymerase chain reaction; rapid diagnosis; smallpox virus

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.

描述（由申请人提供）：DNA 的聚合酶链式反应 (PCR) 扩增是快速检测病原体的最可靠方法 PCR 过程的总体速率由热循环仪和用于酶促的配套生物化学的性能决定。在第一阶段的研究中，已经制造出了一种非常快速的实验性 PCRJet 热循环仪，这种带有电子阀的加压气体机器可以通过热控制+进行 30 个 PCR 循环。 0.2 ¿热循环仪的速度非常快，以至于 PCRJet 已用于在 3.5 至 8.5 分钟内使用 30-40 个 PCR 循环来诊断炭疽芽孢杆菌和蜡状芽孢杆菌。实时检测光学器件，应该可以在不到 8 分钟内常规扩增和检测特殊病原体。在第二阶段研究中，将制造高性能 PCRJet。该机器将能够进行高速 DNA 扩增和基于荧光染料的检测，因此可以使用生化工程方法在约 5 分钟内检测到诊断性炭疽杆菌和天花主要 DNA 片段。为了优化 PCR 过程，将配制非常快速的耐热喷射聚合酶混合物（KOD Pol + dUTPase + PPase），以便在 72¿ 时延伸率 >500 nt/sec C 是可以实现的。PCRJet 热循环仪与改进的 DNA 高速扩增酶学相结合，将能够在不到 2 分钟的时间内诊断生物恐怖主义分子。