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U of Washington, GE Spearhead Effort to Develop Isothermal Amplification-based MDx for Point of Care

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A public-private research team spearheaded by GE Global Research and the University of Washington is six months into a $9.6 million, 18-month grant from the Defense Advanced Research Projects Agency to develop an integrated, handheld molecular diagnostic platform for use at the point of care and in both industrialized and resource-limited settings.

The platform, which will combine isothermal amplification of nucleic acids with paper-based visual detection, is "rapidly approaching" the prototype phase with a first assay for methicillin-resistant Staphyloccocus aureus, scientists involved with the project told PCR Insider recently.

When completed, the device is expected to enable a diagnosis from raw sample in less than an hour, and eventually may also be able to run molecular assays for influenza, sexually transmitted infections, and other infectious diseases, the researchers said.

The public-private team, which also comprises researchers from Epoch Biosciences, Seattle Children's, and PATH, first convened in 2011 under a previous $4 million DARPA-funded project led by UW bioengineer Paul Yager to develop an instrument-free nucleic acid amplification system for pathogen identification.

Having identified and vetted various component technologies, the group this spring was awarded a Phase I DARPA grant, which is essentially the second phase following the successful aforementioned "base period," said Joseph Suriano, a technology leader in engineered materials, chemistry, and physics at GE Global Research in Niskayuna, NY.

"We had previously demonstrated individual components of an integrated system, and now are rapidly approaching the ability to integrate all of those components in a single prototype device, and are pretty excited about the progress we're making there," Suriano said. The group hopes to demonstrate a "fully working prototype" at a meeting with DARPA later this month, he added.

"This whole device is going to have to integrate sample prep with isothermal amplification and downstream detection," said Scott Duthie, a molecular biologist at GE Global Research. "We want it at a point where the user will put a nasal swab in and push a button, and somewhere between 45 minutes to an hour later, your answer will appear. It will actually be a … detection line, much like a pregnancy test, that you'll take a picture with using a cell phone, and that will get [sent] to a physician somewhere who will do a diagnosis."

For isothermal amplification, the group is using a technology called isothermal strand displacement amplification, or iSDA, from Epoch, a division of international diagnostic firm EliTech Group.

The iSDA method comprises "a DNA polymerase expansion, then a nick by an endonuclease, which opens up a gap in the DNA that the polymerase can recognize," Duthie said. "So you get repeated rounds of extension from a primer, nicking by the endonuclease, and extension from the primer again … and build up an amplicon base that you can then detect relatively fast. With iSDA we're talking maybe 20- to 30-minute amplification from as little as one to two copies of DNA input."

The platform's detection capabilities will be powered by disposable "2D paper networks" developed primarily in the Yager lab at UW. This technology is similar to, but not exactly the same as, lateral flow technology, which is currently used in multiple point-of-care diagnostic assays either on the market or under development. According to the Yager lab website, although the 2DPN technology is comparable in cost and ease of use to these conventional lateral flow tests, it may provide greater sensitivity and multiplexing capabilities.

While the group is confident in the performance of the nucleic acid amplification and detection technologies, sample prep — a common bugaboo of many point-of-care molecular diagnostic platforms — remains a challenge.

"Sample prep at this time is going to be a nasal swab … [but] we've got to find the right swab to use," Duthie said. "We want it to absorb liquids and release organisms very readily. Sample prep is problematic because we've got to get rid of cell debris and other inhibitors within the cell for isothermal amplification. One of the big questions we have is: Can we move the genomic DNA around to where we need it on the lateral flow strip to actually do the amplification?"

Duthie added that UW is currently investigating a liquid-phase sample prep method, while GE is looking into membranes and solid-phase approaches akin to well-known Whatman FTA technology for nucleic acid collection, storage, and purification.

"We're trying to get the best sample prep down," Duthie said. "If you think about trying to detect one or two organisms [in a sample], you've really got to have good cell lysis to get all the DNA out of there so it can be amplified. I think the collaboration between GE and UW will get this finalized."

The team's first assay will be for MRSA, with the rationale that this antibiotic-resistant bacterium is especially problematic in institutional settings where people live in close quarters, such as hospitals, prisons, and military bases — hence the involvement of DARPA.

However, the group hopes to develop a platform with interchangeable components that would allow testing for other pathogens, including STDs and various viruses such as influenza with RNA as the genetic material.

The researchers, particularly Yager, are involved in other projects to develop diagnostic devices, many of which may use some of the same component technologies. For instance, some members of the team are developing a point-of-care immunoassay device for influenza detection under a $5.7 million grant from the National Institutes of Health.

Further, GE Global Research and partner InDevR recently completed a $5.8 million DARPA-funded project to develop rapid, point-of-care testing devices for flu; while a group comprising researchers from UW, PATH, Epoch, and Sony's Micronics was previously awarded $15.4 million from the Bill & Melinda Gates Foundation to develop a portable diagnostic device for the developing world.

From GE's standpoint, the collaborative efforts have been highly productive so far, even if it may still be months or years before a commercial platform is ready for deployment.

"We've got an outstanding team that is working very well together – better than any I've ever seen," Suriano said. "Everybody brings unique … complementary things, and there are a couple of areas where we have competing technologies, but people are just trying to find the best technology for this fully integrated device and get it out there as quickly as we can."

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