NEW YORK (GenomeWeb) – Amid the growing Ebola outbreak in West Africa, a pair of University of Arizona researchers are working to adapt a novel PCR technology for a hand-held test that can detect the virus more rapidly and efficiently than existing molecular diagnostics.
According to U of A investigators David Galbraith and Jeong-Yeol Yoon, the PCR approach has already shown promise in early-stage testing for bacterial pathogen detection. With the help of a one-year National Science Foundation grant, they now aim to optimize the technology for screening viral infections.
And while Ebola is their initial focus, they believe that their test can ultimately be optimized for the detection of any biological disease organism, including ones that impact the agricultural industry.
PCR is a widely used and highly sensitive technology for infection screening, and in the last few months the US Food and Drug Administration has issued Emergency Use Authorization to several real-time RT-PCR tests for detecting Ebola in human blood specimens, including one from Roche last month.
But standard PCR requires large and expensive instrumentation typically available only in laboratories. Additionally, when considering an urgent infection such as Ebola, standard PCR can be relatively slow to provide answers, complicating the clinical decision-making process.
To address these limitations, Yoon has developed a technology called droplet-on-thermocouple silhouette quantitative PCR, or DOTS qPCR, which is serving as the foundation of a "sample-to-answer" portable device that handles all aspects of the analysis process in as little as five minutes.
"We don't need to have the laboratory environment … but can actually bring our instrumentation with us wherever and whenever," he told GenomeWeb.
The key to DOTS qPCR, Yoon explained, is its novel approach to thermal cycling. In traditional PCR, a sample is placed in a tube that is then put into chamber that is heated and cooled in order to facilitate DNA amplification.
In Yoon's approach, a patient sample is put in a water droplet instead of a test tube. The droplet is then placed in an oil suspension within the handheld device where it travels through hot and cold chambers, eliminating the time required to heat up and then cool down a single chamber as with standard PCR.
Further, DOTS qPCR samples do not require extensive purification because cellular debris accumulates at the water/oil interface while nucleic acids remain in the water droplet, making the approach even more efficient and more amenable to a point-of-care setting.
Lastly, Yoon's PCR method does not require fluorescent reporters to track the amplification process, which is instead done by monitoring the shape of the water droplet as interfacial tension changes its shape during the thermal cycling process.
Yoon said that patent applications have been filed on DOTS qPCR and that he has submitted a manuscript describing the technology to a peer-reviewed publication, but that neither has yet published. In 2012, Yoon published data related to a predecessor verison of the technology.
After successfully testing a prototype of their handheld DOTS qPCR device with bacteria and having received $200,000 from the NSF, Yoon and Galbraith are now starting work on adapting it for Ebola detection. Since Ebola is a single-stranded RNA virus, this effort will largely involve the incorporation of a reverse-transcription step to enzymatically convert RNA to DNA prior to amplification.
Once that is done, proof-of-concept work will begin in earnest — but not with Ebola itself due to the safety issues and handling requirements. Instead, the investigators will use plant viruses, including one that affects citrus trees that Galbraith said has a genome nearly the same length as the Ebola virus.
Meanwhile, the researchers are also trying to refine the prototype device into one that is more portable and appropriate for use in the field, and potentially can be coupled with a smartphone for data processing and result communication.
"Once we've established the instrument works … we would need people within Ebola facilities in the United States and see … if we can get them to test it out," Galbraith said.
Should the DOTS qPCR test prove successful for Ebola, Yoon and Galbraith see far-ranging potential for the technology since it should theoretically enable the detection nucleic acids associated with any virus.
And because it does not require samples to be sent to a lab for purification and analysis, it could be deployed almost anywhere. For instance, "you might be able to screen [airline] passengers during the boarding process, and then be able to filter them for further testing" upon positive results, Galbraith said.
He added that he has also received interest in the technology from people in the farming industry.
"In production agriculture, there are a lot of emerging viruses you encounter," he said. "For someone to be able to collect samples directly from plant leaves and put them in a device to measure whether a particular virus is present is very attractive."
Getting the technology to market, however, may be challenging, Galbraith cautioned.
Because there are no consumables associated with the device, he said he is wary about whether instrument manufacturers would be interested in its commercialization.
"Manufacturers love to sell the fluorescent nucleotides that are used for detection purposes," and there is the possibility a company may acquire the patents on the system only to shelve it, he said.
Galbraith and Yoon have also contemplated starting a company around the DOTS qPCR technology, but given the urgency of the Ebola epidemic, they are staying focusing on moving it along under the NSF grant project for now.