University of Arizona scientists have developed a "wire-guided" droplet-based PCR method that they claim can amplify and detect gene targets in real time in about three minutes.
With the aid of a state-funded technology transfer program, the scientists are incorporating a company tentatively called Fast PCR Diagnostics to cultivate the technology into an ultrafast, portable system for diagnosing blood infections in emergency room settings.
In addition, the group was recently awarded a contract for an unspecified amount from the South Korean government to adapt the technology for use in veterinary diagnostic applications, such as rapid, field-based testing of foot-and-mouth disease, Jeong-Yeol Yoon, lead inventor of the technology, told PCR Insider this week.
The new technology makes use of 10-µL pendant droplets on the ends of syringe needles and containing both sample and necessary PCR reagents. The researchers can "centrifuge" these droplets to concentrate sample using a vibration motor under pulse control. Then, under robotic control, the needle tip moves the droplets through a superhydrophobic silicon oil matrix and three constant temperature zones, which serves to both rapidly extract and amplify DNA from the sample.
"Our technology is based on … a PCR mixture hanging on the end of a needle … that is moving around three different heat chambers connected by tunnels," Yoon said. "Each chamber maintains a [constant] temperature. So you're not really heating up and cooling down. The heating and cooling should then be extremely fast. In most other PCR technology, however fast they are claimed to be, they still use conduction as their heat transfer mechanism. In our case, it's convection. The heat transfer coefficient for convection is much higher than conduction, so the key here is actually the convection."
Yoon added that immersing the sample- and reagent-containing droplets in the silicon oil serves to purify DNA for amplification because "the proteins on the cells in the sample matrix tend to go toward the silicon oil and water interface, but all the other PCR primers and genomes stay inside the core of the droplet, so we actually achieve a sort of separation."
Typically, he noted, PCR amplification is hindered by a "dirty" sample matrix, "but our system actually works better if the sample is dirty."
Yoon and colleagues described the development and early performance of their method in papers published in 2010 in the journal Faraday Discussions, and this September in the Journal of Biological Engineering.
In the first paper the scientists demonstrated the use of the method to detect 2009 H1N1 influenza A gene sequences; and in the JBM paper they showed how it could be used to amplify the 1,500-bp peptidase D sequence from Escherichia coli samples. In the most recent paper, the group reported a total assay time of 23 minutes, including droplet centrifugation, DNA extraction, and approximately 10 minutes of thermal cycling.
Yoon said this week, however, that the group has since reduced this thermal cycling time to about three minutes, which would reduce the total assay time to around 15 minutes.
In addition, Yoon and colleagues have gradually reduced the size of their system, from a breadboard model taking up the entire surface of a lab bench to its current iteration, about the size of a desktop computer tower. He said that the researchers believe they can further reduce the size to that of a laptop computer, making the device portable for use in near-patient settings.
"I'm not saying it will be handheld, but hopefully within a couple of years we can deliver a portable device that is battery operated and does not really need an external computer," Yoon said. "And it is cartridge-based, so you can … [perform an assay] once and throw it away to avoid cross contamination. That's what we are envisioning."
What's more, to this point the researchers have confirmed the results of their rapid PCR using gel electrophoresis analysis of end products. However, they also plan to enable their device to perform real-time PCR, and believe they have unearthed an extremely economical optical detection system to do so.
"We initially tried to use optical fibers, et cetera, and then realized it was too expensive," Yoon said. "So we purchased the iPhone microscope" — an after-market accessory that attaches to Apple's iPhone and turns the camera into a 10X microscope. "It's only [about $40] on the market, and we can patch that iPhone microscope into our tunnels and monitor the fluorescence of SYBR Green dye," Yoon added. "We do have some data on the quantification and it works pretty great. We were quite amazed. Everybody has a smart phone. All you need is to download the app, attach the microscope lens to your system, and that's our optical detection system."
Although the technology has many potential applications, Yoon and colleagues wish to focus first on using it to potentially diagnose blood infections in an acute hospital care setting. "Hopefully doctors can … take a sample from blood, and then use this system to run PCR right away, get the results in less than 10 minutes, and then make a decision … [regarding] the proper dose and type of antibiotics. That is a major and immediate goal at this point," he said.
The UA tech transfer office has helped the group incorporate its startup, Fast PCR Diagnostics, and last month the nascent company was one of 10 startups selected from more than 50 statewide applicants to participate in the AZ Furnace Accelerator, a statewide, public-private partnership among the Arizona Commerce Authority, BioAccel, Arizona State University, Northern Arizona University, University of Arizona, Thunderbird School of Global Management, Arizona Technology Enterprises, and Dignity Health Arizona.
As part of its participation in the program, Fast PCR Diagnostics received $25,000 in funding from the Arizona Commerce Authority and BioAccel, and will be provided incubator office space near the UA campus to further develop and commercialize the technology for blood infection testing.
Meantime, Yoon's lab has received a contract from South Korea to develop veterinary diagnostic tests using the platform in the wake of last year's nationwide outbreak of foot-and-mouth disease. According to Yoon, standardized antibody-based foot-and-mouth disease testing did not provide a fast enough turnaround time to prevent the disease from rapidly spreading, causing the country to kill approximately one-third of its cattle herd.
"If we could run portable PCR on the spot right away, we could prevent that from happening," Yoon said.
The group about a year ago filed a PCT patent application covering its technology, and plans to soon file another patent covering recent modifications and upgrades, after which, Yoon said, it hopes to publish another paper describing these recent breakthroughs in more detail.