Helicase-based rapid DNA diagnostic for Biodefense

基于解旋酶的生物防御快速 DNA 诊断

• 批准号: 6992501
• 负责人: Huimin Kong
• 金额: $ 46.99万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2005
• 资助国家: 美国
• 起止时间: 2005-07-01 至 2007-06-30
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/6992501
• 关键词: Bacillus anthracis; DNA directed DNA polymerase; DNA primers; DNA virus; Ebola virus; Marburg virus; RNA directed DNA polymerase; RNA virus; biohazard detection; biotechnology; bioterrorism /chemical warfare; communicable disease diagnosis; diagnosis design /evaluation; environmental toxicology; helicase; nucleic acid amplification techniques; nucleic acid chemical synthesis; portable biomedical equipment; rapid diagnosis; temperature

DESCRIPTION (provided by applicant): Nucleic acid based diagnostic devices allow accurate identification of biological agents by specifically amplifying specific target sequences from environmental samples. Current diagnostic tests rely on PCR, a more costly and time-consuming detection method that cannot be easily adapted for use in the field. An inexpensive, easy-to-use hand-held diagnostic test for the detection of biological agents onsite will allow first responders to rapidly assess a potential bioterrorism threat. Our laboratory has developed an isothermal DNA amplification system that can be performed independently of a thermal cycler. The technology, Helicase-Dependent Amplification (HDA), employs helicase to enzymatically unwind DNA, rather than using high temperature, to expose the target sequence and allow specific primers to anneal, followed by DNA polymerase mediated primer extension. Products from each cycle of HDA enter the next cycle, resulting in exponential amplification. The technology has been demonstrated to detect as little as 100 cells of the bioterrorism agent Bacillus anthracis. With continued research, the sensitivity and efficiency of HDA will be improved upon and used in the next generation of isothermal nucleic acid diagnostic tests. The main objective of the Phase I research is to innovate HDA by determining and addressing the limitations of the HDA platform and to develop a reverse transcription HDA (RT-HDA) system for use in a multiplexed test for the simultaneous detection of DNA and RNA encoded pathogens, such as B. anthracis and Ebola virus. The aims of Phase I will examine devising an efficient primer design protocol, improving HDA by enhancing the performance of HDA enzymes and developing a one-step, isothermal RT-HDA platform. Innovations to the HDA technology in Phase I will allow the development and production of hand-held HDA based diagnostic devices in Phase II. By innovating the quality of diagnostic testing, we seek to provide emergency personnel with the necessary tools to protect communities from potential biological threats.

描述（由申请人提供）： 基于核酸的诊断设备可以通过特异性扩增环境样品的特定靶序列来准确鉴定生物学剂。当前的诊断测试依赖于PCR，这是一种更昂贵，更耗时的检测方法，无法轻易适应该现场。一项廉价，易于使用的手持诊断测试，可在现场检测生物剂，将使急救人员能够快速评估潜在的生物恐怖威胁。我们的实验室已经开发了一个等温DNA扩增系统，该系统可以独立于热循环仪进行。该技术（依赖性酶依赖性扩增（HDA））采用解旋酶在酶上放松DNA而不是使用高温来暴露靶序列并允许特定的引物退火，然后进行DNA聚合酶介导的引物扩展。 HDA周期中的产品输入下一个周期，从而导致指数扩增。该技术已被证明可检测到生物恐怖剂的100个细胞。通过持续的研究，将在下一代等温核酸诊断测试中提高HDA的敏感性和效率。第一阶段研究的主要目的是通过确定和解决HDA平台的局限性来创新HDA，并开发用于多路复用测试的逆转录HDA（RT-HDA）系统，以同时检测DNA和RNA编码的病原体，例如B. annthracis和b。anthracis和ebola virus。第一阶段的目的将研究设计有效的底漆设计协议，并通过增强HDA酶的性能并开发一步等温线RT-HDA平台来改善HDA。 I阶段对HDA技术的创新将允许在第二阶段的基于HDA的诊断设备的开发和生产。通过创新诊断测试的质量，我们试图为紧急人员提供保护社区免受潜在生物学威胁的必要工具。