Fast and Simple Real Time PCR for Quantitative Molecular Diagnostic Testing

描述（由申请人提供）：真实的时间定量PCR正迅速取代常规或“终点”PCR作为主要诊断工具。Thermal Gradient Inc. (TG)已经表明，基于其专有技术的微流体设备可以快速简单地进行生物防御应用的终点PCR。“快速”是指反应在几分钟内完成，而不是像传统设备那样在几小时内完成。TG希望将其技术的优势扩展到定量诊断测试中。拟议的项目，快速和简单的真实的时间PCR诊断测试，旨在建立的可行性进行“真实的时间”PCR比传统的手段更迅速，更方便。这里设想的产品将是一种紧凑的仪器，可以在手术室或床边使用，以确定体液中的微生物负荷或活检中的肿瘤侵袭程度，所有这些都在样本采集后30分钟内完成。除了快速、实时PCR热循环外，实现这一目标所需的所有技术今天都存在。在用于生物防御的“终点”PCR中，反应进行至完成，然后使用荧光检测来从扩增的DNA中识别在许多可能的病原体中已经用于攻击的病原体。在“实时”PCR中，目标是从反应速率确定通常一种病原体或突变序列的起始浓度，尽管也可以检测多个靶标。这需要在进行PCR时对反应混合物进行荧光检测。在第一阶段的申请中，我们建议实现两个目标：1。表明TG装置的“实时”版本，即允许在样品混合物流过装置时观察样品混合物的版本，仍将进行“终点”PCR;以及2.表明预期设计良好的检测系统将能够检测到将由“实时”设备发出的荧光信号是合理的。如果这些目标得到满足，则很可能在II期项目中成功地进行真实的时间PCR，该项目旨在展示非常快速、完整和定量的分子诊断系统的所有部分。

DESCRIPTION (provided by applicant): Real time quantitative PCR is rapidly replacing conventional or "end-point" PCR as a major diagnostic tool. Thermal Gradient Inc. (TG) has already shown that microfluidic devices based on its proprietary technology can carry out end-point PCR quickly and simply for bio-defense applications. "Quickly" is understood to mean reactions completed in minutes instead of hours, as with conventional equipment. TG would like to extend the benefits of its technology into quantitative diagnostic testing. The proposed project, Fast and Simple Real Time PCR for Diagnostic Testing, is intended to establish the feasibility of performing "real time" PCR more rapidly and conveniently than conventional means. The product envisioned here would be a compact instrument that could be used in the operating room or at the bedside to determine, among other applications, the microbial load in body fluids or the extent of tumor invasion in a biopsy, all within 30 minutes of sample collection. All of the technology required to carry this out exists today except for rapid, real-time PCR thermal cycling. In "end-point" PCR for bio-defense, the reaction is carried out to completion and then fluorescent detection is employed to identify from the amplified DNA which among many possible pathogens have been employed in an attack. In "real-time" PCR the objective is to determine from the rate of the reaction the starting concentration of usually one pathogen or a mutant sequence, although detection of multiple targets is also possible. This requires fluorescent detection of the reaction mix while it is undergoing PCR. In this Phase I application, we propose to achieve two aims: 1. Show that a "realtime" version of the TG device, one that permits observing the sample mix as it flows through the device, will still carry out an "end-point" PCR; and 2. Show that it is reasonable to expect that a well designed detection system will be able to detect the fluorescent signal that will be emitted by the "real-time" device. If these goals are met it is very likely that real time PCR can be carried out successfully in a Phase II project meant to demonstrate all the pieces of a very fast, complete, and quantitative molecular diagnostic system.