CASCADE ROLLING CIRCLE AMPLIFICATION FOR DNA DIAGNOSTICS

目前用于检测致病原的多种核酸扩增系统 试剂可能不会产生一致的结果，因为最佳扩增 每个靶序列的条件都不同。梯级滚动圈 扩增(CRCA)是一种新型的等温扩增系统，它使用 扩增环状探针而不是靶标的通用引物。它 快速、高度灵敏、易于多路传输、持续产出 在一小时内放大超过十亿倍，而不使用 热循环仪。如果其中一个引发物是能量转移标记引发物， CRCA产物的均相荧光检测在 一个封闭的系统。化验的速度和简便性使这项技术 适用于发展中国家的实地工作或使用，以及 诊断应用中更简单的自动化。这样做的可行性 该系统已在初步实验中得到验证。第一阶段 目标包括连接和扩增条件的优化 以实现最大的信号强度和灵敏度，并优化 能量转移标记的底物结构，产量最高 不影响放大的荧光信号背景比 效率。建议的方法的速度和灵敏度将是 与使用致病靶标II期的传统技术相比 研究将集中在该系统的临床应用上。 建议的商业应用： 这种扩增和检测系统可以构成任何DNA的基础 诊断程序，其中特定的核的存在或不存在 并确定靶序列，例如传染病病原体， 遗传性种系突变或体细胞突变在癌症筛查中的应用 测试这种分析是高度敏感、快速和简单的，我们估计 这将比目前的方法成本更低。因为它可以被执行 在封闭的系统中，假阳性是由残留污染造成的 将被最小化。该系统易于实现简单的自动化。

Many current nucleic acid amplification systems for detecting pathogenic agents may not yield consistent results because optimal amplification conditions vary with each target sequence. Cascade rolling circle amplification (CRCA) is a novel isothermal amplification system that uses generic primers to amplify circularized probes instead of the target. It is rapid, highly sensitive, and easily multiplexed, consistently yielding over a billion-fold amplification within one hour without using a thermocycler. If one of the primers is an energy transfer-labeled primer, homogeneous fluorescence detection of the CRCA product is possible within a closed system. The speed and ease of the assay make this technology suitable for field work or use in developing countries, as well as for simpler automation in diagnostic applications. The feasibility of this system has been demonstrated in preliminary experiments. Phase I objectives include optimization of ligation and amplification conditions to achieve maximum signal strength and sensitivity, and optimization of energy transfer-labeled primer structures that yield the highest fluorescence signal to background ratio without compromising amplification efficiency. The speed and sensitivity of the proposed method will be compared with traditional techniques using a pathogenic target Phase II studies will focus on clinical applications of this system. Proposed commercial applications: This amplification and detection system can form the basis of any DNA diagnostic procedure where the presence or absence of a specific nucleic and target sequence is determined, such as an infectious disease agent, inherited germline mutation, or somatic mutation in a cancer screening test The assay is highly sensitive, fast and simple, and we estimate that it will be less costly than current methods. Because it can be performed in a closed system, false-positive results from carry-over contamination will be minimized. The system is readily amenable to simple automation.