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

RAPID：开发埃博拉手持式快速分子诊断工具

• 批准号: 1511093
• 负责人: David Galbraith
• 金额: $ 20万
• 依托单位: University of Arizona
• 依托单位国家: 美国
• 项目类别: Standard Grant
• 财政年份: 2015
• 资助国家: 美国
• 起止时间: 2015-01-01 至 2016-12-31
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=1511093&HistoricalAwards=false
• 关键词: RAPID; Development; handheld; rapid; molecular

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.

1511093 Galbraith, David W. 亚利桑那大学埃博拉病毒感染目前引起了相当大的关注，因为缺乏有效的干预措施、其高致死率以及缺乏快速、灵敏的方法来检测病毒的存在。 分子诊断基于使用诺贝尔奖获得者卡里·穆利斯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，代表了一种敏感的感染筛查方法。然而，当前的 PCR 仪器体积庞大、昂贵且复杂，并且无法以做出临床决策所需的速度运行。 研究人员开发了一种新型 PCR 设备，称为 Droplet-On-Thermo Couple Silhouette 定量 PCR (DOTS qPCR)，该设备基于感测界面效应而不是现有的 PCR 反应荧光检测进行操作。这种创新的检测方法可大大加快分析速度，从样品到结果的时间只需 5 分钟之内。该 RAPID 项目旨在进一步开发该设备，以展示其从人体样本中检测埃博拉病毒的适用性，将该设备与标准智能手机连接以进行数据收集、处理和传输，并展示如何将其用作手持设备。除了快速检测埃博拉病毒外，DOTS-PCR 设备还将普遍适用于检测与美国国家健康、农业和安全有关的任何生物疾病微生物。 新颖的 PCR 方法 (DOTS qPCR) 首次利用接口上的创新工程原理来实现液滴驱动、抑制解除和 PCR 反应的传感，从而使样本到应答时间缩短至 5 分钟。为了诊断埃博拉病毒是否存在，研究人员建议证明可重复性、病毒种类的区分、亚皮克检测限以及在存在血液/组织污染物的情况下 28 秒/周期的热循环速度。该项目的具体目标是 (i) 开发适合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) 集成智能手机使用界面效应而不是传统的荧光传感来识别扩增子，并开发适当的数据处理和结果通信软件。尽管该技术旨在检测埃博拉病毒，但该技术显然在其他特定生物体和制剂的高通量、低成本检测中具有普遍适用性。支持这些目标的智力优势在于创新地利用了水性反应液滴表面和周围油相的界面效应，该界面效应与污染蛋白质的分配和扩增产物的形成相关，从而导致检测速度远远优于传统的基于荧光的方法。仪器。

项目成果

Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

利用界面效应实现低阈值循环检测和抑制缓解的创新 qPCR

• DOI: 10.1126/sciadv.1400061
• 发表时间: 2015-09
• 期刊: Science Advances
• 影响因子: 13.6
• 作者: Harshman DK;Rao BM;McLain JE;Watts GS;Yoon JY
• 通讯作者: Yoon JY