RAPID: Development of a handheld, rapid molecular diagnostic tool for Ebola

1511093 Galbraith, David w .亚利桑那大学由于缺乏有效的干预措施，其高致死率以及缺乏快速和敏感的检测病毒存在的手段，埃博拉病毒感染目前引起了相当大的关注。利用诺贝尔奖获得者Cary Mullis在美国开发的聚合酶链反应（PCR）技术检测埃博拉病毒基因组的分子诊断是筛查感染的一种敏感方法。然而，目前的PCR仪器体积大、价格昂贵且复杂，而且不能以做出临床决策所需的速度运行。研究人员开发了一种新的PCR装置，称为热电偶上的液滴剪影定量PCR (DOTS qPCR)，该装置基于传感界面效应而不是现有的荧光检测PCR反应。这种创新的检测方法可以在5分钟内实现更快的分析时间。RAPID项目旨在进一步开发该设备，以证明其适用于从人体样本中检测埃博拉病毒，将该设备与标准智能手机连接，用于数据收集、处理和传输，并展示如何将其用作手持设备。除了埃博拉病毒的快速检测，DOTS-PCR设备将普遍适用于检测任何与美国国家卫生、农业和安全有关的生物疾病有机体。新的PCR方法（DOTS qPCR）首次在界面上利用创新的工程原理来实现液滴驱动、抑制解除和PCR反应的感知，从而使样品到答案的时间短至5分钟。为了诊断埃博拉病毒的存在，研究人员建议在存在血液/组织污染物的情况下证明可重复性、病毒种类的分化、亚图检测限以及28秒/循环的热循环速度。该项目的具体目标是(i)开发适用于诊断用途的第二代手持式DOTS qPCR， （ii）展示在存在典型血液/组织污染物的情况下识别目标的能力，（iii）集成基于智能手机的扩增子识别，使用界面效应而不是传统的荧光传感，并开发用于数据处理和结果通信的适当软件。虽然这项技术的目的是检测埃博拉病毒，但显然在其他特定生物有机体和病原体的高通量、低成本检测中具有普遍适用性。支持这些目标的知识价值在于创新地利用了水反应滴表面和周围油相的界面效应，这些界面效应与污染蛋白质的分配和扩增产物的形成有关，从而使检测速度远远优于传统的基于荧光的仪器。

1511093 Galbraith, David W.University of Arizona Ebola virus infection provides considerable current cause for concern due to a lack of effective interventions, its high lethality, and a lack of rapid and sensitive means to detect viral presence. Molecular diagnostics, based on detection of the Ebola virus genome using the Polymerase Chain Reaction (PCR) technique developed in the US by Nobel Laureate Cary Mullis, represents a sensitive approach to screen for infection. However, current PCR instruments are large, expensive and complicated, and do not operate at the speeds required to make clinical decisions. The investigators have developed a novel PCR device, termed Droplet-On-Thermocouple Silhouette quantitative PCR (DOTS qPCR), which operates on the basis of sensing interfacial effects instead of the established fluorescence detection of the PCR reaction. This innovative detection method enables much faster analysis times with sample-to-answer times within 5 minutes. This RAPID project aims to further develop the device to demonstrate its applicability for detection of Ebola virus from human samples, to interface the device with standard smartphones for data collection, processing and transmission, and to show how it can be used as a handheld device. Beyond the rapid detection of Ebola virus, the DOTS-PCR device will be universally applicable for detection of any biological disease organism of concern to the US National health, agriculture, and security. The novel PCR methodology (DOTS qPCR) utilizes for the first time innovative engineering principles on interfaces to achieve droplet actuation, inhibition relief, and sensing of the PCR reaction, resulting in sample-to-answer times as short as 5 minutes. Towards diagnosis of the presence of Ebola virus, the investigators propose to demonstrate reproducibility, differentiation of virus species, sub-picogram limit of detection, and thermocycling speeds of 28 s/cycle in the presence of blood/tissue contaminants. The specific aims of this project are (i) development of a second-generation handheld DOTS qPCR appropriate for diagnostic use, (ii) demonstration of the ability to identify target in the presence of typical blood/tissue contaminants, (iii) integration of smartphone-based identification of amplicons using the interfacial effect instead of traditional fluorescence sensing, with development of appropriate software for data processing and result communication. Although aimed at detection of Ebola, this technology clearly has general applicability in the high-throughput, low-cost detection of other specific biological organisms and agents. The intellectual merit underpinning these aims rests in the innovative exploitation of interfacial effects on the surface of aqueous reaction droplets and the surrounding oil phase associated with the partitioning of contaminating proteins and the formation of amplification products resulting in detection speeds far superior to traditional, fluorescence based instruments.