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

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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.