Development of a one-step isothermal DNA amplification system for diagnostics

开发用于诊断的一步式等温 DNA 扩增系统

• 批准号: 7111405
• 负责人: Huimin Kong
• 金额: $ 12.62万
• 依托单位: BIOHELIX CORPORATION
• 依托单位国家: 美国
• 财政年份: 2006
• 资助国家: 美国
• 起止时间: 2006-04-11 至 2006-10-10
• 项目状态: 已结题
• 来源: https://reporter.nih.gov/project-details/7111405
• 关键词: DNA; biohazard detection; biotechnology; diagnosis design /evaluation; diagnostic tests; helicase; nucleic acid amplification techniques; rapid diagnosis; technology /technique development; thermostability

DESCRIPTION (provided by applicant): Molecular diagnostic tests that rely on the amplification of specific sequences to identify genes of interest frequently use the polymerase chain reaction (PCR). To perform PCR, sensitive and, often, expensive instrumentation is required for thermocycling, which facilitates heat denaturation, primer annealing and primer extension by a thermostable DNA polymerase. The equipment requirement for performing PCR limits its portability and use in point-of-need diagnostics, for example in the field to analyze potential bioterrorism agents or at a patient's bedside. Thus, there is a need for an easy to use, isothermal DNA amplification system that can easily be used to perform diagnostic tests in the field. In the proposed research, we describe a one-step Helicase-Dependent amplification system based on a new HDA platform: extreme- thermophilic HDA or eHDA system. The new eHDA platform will allow assays to be performed in the presence of all reaction components without any interruptions or possible cross contamination to add additional reagents. A one-step HDA assay will facilitate the development of isothermal analytic specific reagents and other types of diagnostic tests to be used in clinical diagnostic laboratories. The phase I research will examine the feasibility of preparing the eHDA system for use in diagnostic tests, which will be addressed by two specific aims. The first specific aim will address primer design concerns to improve eHDA performance and detection sensitivity. The second specific aim will address increasing eHDA amplification yields by supplementing reactions with accessory proteins. Accessory proteins, such as SSB proteins and MutL, will be purified and tested in eHDA to determine improvements on amplification specificity and sensitivity. The final specific aim will examine optimal detection methods for use with eHDA assays. An appropriate detection system has the potential to improve the sensitivity of the assay beyond that which is observed by standard gel electrophoresis. Results from Phase I research will facilitate development of one- step, isothermal diagnostic tests based on the eHDA platform.

描述（由申请人提供）：依赖于扩增特定序列以识别感兴趣基因的分子诊断检测通常使用聚合酶链反应（PCR）。为了进行PCR，热循环需要敏感且通常昂贵的仪器，这有助于热变性、引物退火和通过热稳定DNA聚合酶的引物延伸。进行PCR的设备要求限制了其便携性和在需要点诊断中的使用，例如在现场分析潜在的生物恐怖主义制剂或在患者床边。因此，需要一种易于使用的等温DNA扩增系统，其可以容易地用于在现场进行诊断测试。在所提出的研究中，我们描述了基于新的HDA平台的一步解旋酶依赖性扩增系统：极端嗜热HDA或eHDA系统。新的eHDA平台将允许在所有反应组分存在的情况下进行测定，而不会出现任何中断或可能的交叉污染以添加额外的试剂。一步HDA测定将促进等温分析特异性试剂和其他类型的诊断测试的发展，以用于临床诊断实验室。第一阶段研究将审查准备eHDA系统用于诊断测试的可行性，这将通过两个具体目标来解决。第一个具体目标将解决引物设计问题，以提高eHDA性能和检测灵敏度。第二个具体目标将通过补充辅助蛋白的反应来解决增加eHDA扩增产率的问题。辅助蛋白，如SSB蛋白和MutL，将在eHDA中纯化和测试，以确定扩增特异性和灵敏度的改善。最终的具体目标将检查用于eHDA检测的最佳检测方法。适当的检测系统有可能提高测定的灵敏度，使其超过标准凝胶电泳所观察到的灵敏度。第一阶段研究的结果将促进基于eHDA平台的一步等温诊断测试的开发。