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）：依赖于特定序列扩增来识别感兴趣基因的分子诊断测试经常使用聚合酶链反应（PCR）。要执行PCR，敏感且通常需要昂贵的仪器来进行热循环，这有助于通过热稳定DNA聚合酶促进热变性，底漆退火和底漆延伸。执行PCR的设备需求限制了其可移植性并在需要的诊断点上使用，例如在现场分析潜在的生物恐怖剂或患者的床边。因此，需要一个易于使用的等温DNA扩增系统，可以轻松地用于在现场进行诊断测试。在拟议的研究中，我们描述了基于新的HDA平台的一步旋转酶依赖性放大系统：极端热嗜热HDA或EHDA系统。新的EHDA平台将允许在所有反应组件的情况下进行没有任何中断或可能的交叉污染以添加其他试剂。单步HDA测定法将促进在临床诊断实验室中使用的等温分析特定试剂和其他类型的诊断测试。第一阶段的研究将研究准备EHDA系统用于诊断测试的可行性，这将由两个特定的目的解决。第一个具体目的将解决引物设计问题，以提高EHDA性能和检测灵敏度。第二个特定目的将通过补充辅助蛋白的反应来解决EHDA扩增产量的增加。辅助蛋白（例如SSB蛋白质和MUTL）将在EHDA中进行纯化和测试，以确定放大特异性和灵敏度的改善。最终的特定目的将检查与EHDA分析一起使用的最佳检测方法。适当的检测系统具有提高测定的灵敏度，超出了标准凝胶电泳观察到的测定法。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。