By Ben Butkus
A team of scientists from the University of Alberta and the Alberta Provincial Laboratory for Public Health has published research demonstrating the ability of its integrated, multiparameter, gel array-based PCR assay to detect herpes simplex virus from clinical samples with accuracy comparable to that of conventional PCR.
The research serves as important proof of principle to support the efforts of University of Alberta spinout Aquila Diagnostic Systems, which is attempting to commercialize the platform.
Although Aquila is not currently developing the technology for sexually transmitted disease testing, the paper is a "really important demonstration" of the platform's ability to do multiparameter molecular testing in the markets Aquila is pursuing – namely, veterinary livestock testing, diagnosis of tropical diseases like malaria, and pharmacogenomic testing, according to a company official.
The group's core enabling technology, called In-Gel, comprises a polyacrylamide gel and PCR reagents photopolymerized in a mold to create an array of semi-solid posts with volumes of less than 1 µL each that serve as reaction vessels for parallel PCR reactions.
The researchers have also created a prototype analysis device consisting of a simple Peltier element for thermal cycling, and an inexpensive laser excitation source and CCD camera for product detection via melting curve analysis.
PCR Insider first reported on the In-Gel technology in September 2010, after the University of Alberta group led by Linda Pilarski published a paper in Analytical Chemistry describing the technology and demonstrating its ability to perform multiple microliter-scale PCR reactions in parallel using externally added nucleic acid template (PCR Insider, 9/9/2010).
Since that time, the researchers have been working on adapting the platform for clinical diagnostic applications. For example, in September team member Stephanie Yanow discussed with PCR Insider her efforts to develop the technology into a point-of-care testing device for diagnosing malaria by amplifying DNA from Plasmodium species directly from whole blood and filter paper samples (PCR Insider, 9/8/2011).
Most recently, in a paper published last month in Lab on a Chip, Pilarski, Yanow, and colleagues have taken the next step toward developing a clinically viable version of the In-Gel technology, by demonstrating its ability to simultaneously detect HSV-1 and HSV-2 DNA together with integrated positive and negative controls from unprocessed genital swabs.
More specifically, the group used a version of In-Gel featuring individual 0.67-µL gel posts to detect HSV DNA templates in as little as 2.5 nL of raw sample. They blindly tested 45 genital swab specimens using the platform, and achieved a concordance of 91 percent with conventional PCR.
"What we've really demonstrated here … is the ability to do multiparameter testing," Pilarski told PCR Insider this week. "That means multiple different tests, in parallel, simultaneously, for the same sample."
Pilarski noted that this is distinct from multiplex PCR testing, which is also a challenge that many commercial entities and academic groups are attempting to address.
"Multiplex is when you might have multiple primers in the same well," Pilarski said. "That's kind of a difficult thing to do. With multiparameter, if you simply have one well with one set of primers, another well with another set of primers, and positive and negative controls also in different wells, then you can do all your tests in one run with one sample."
Pilarski noted that the group achieved these results on an early prototype analysis platform, of which "we have eight different versions, all varying slightly. But even with the constraint of using instruments that are not yet standardized, we got 91 percent concordance with conventional PCR," she said.
"Now we've got an enclosed instrument that has precisely standardized components … and we think we can only get better in terms of concordance with conventional results," she added. "Even so, HSV culture, which is the standard test, is only about 83 percent concordant with conventional PCR."
Aquila, which has licensed the technology from the University of Alberta and which works closely with researchers there to develop applications for the In-Gel platform, will not immediately pursue HSV testing or any other type of STD diagnosis per se, Jason Acker, chief technology officer at the company, told PCR Insider this week.
Nevertheless, the Lab on a Chip paper "really demonstrated the ability of the technology to … test multiple targets on a single sample. We've done that not just for HSV, now, but for a variety of other targets in work that hasn't yet been published."
Acker said that the company is looking at "a variety of applications," but is beginning to focus on three potential markets. First, on the molecular diagnostics side, the company is exploring infectious disease testing, particularly for neglected and tropical diseases such as malaria.
"We're working with Stephanie Yanow and her team on the malaria application, so we are … porting all the work that team has done to the commercial platform with the plan of supporting [its] efforts to really use the technology in Africa and South America," Acker said.
In addition, Aquila has done "a lot of work" in the pharmacogenomic testing arena, "to test for drug resistance, particularly in the area of breast cancer and tamoxifen treatment," Acker said.
These markets, while potentially lucrative, also require a much larger investment of time and money. As such, Aquila is eyeing the more near-term opportunity of veterinary livestock testing.
"We are really developing a lot of momentum in the area of livestock disease testing, looking at disease outbreaks in livestock herds that have a huge impact not only on the treatment of the animals, and the productivity of the animals, but also the biosecurity and safety of the food source," Acker said. "The attraction with veterinary medicine is that there is really a practical need now, and the path to getting the commercial product into the market is much shorter compared to the regulatory challenges of a healthcare diagnostic."
Aquila has refined the instrumentation platform and the disposable chip technology to bring it closer to a mass-producible commercial product, but still has "some engineering challenges to overcome," Acker said. Nevertheless, the company believes that its platform can eventually offer inexpensive, point-of-care testing for a variety of markets.
One of the biggest recent technological developments the group has made toward this goal is to develop a universal sample prep method that will allow users to test a variety of unprocessed samples on the platform.
Pilarski said that the group has not yet disclosed details of this method because it is pending patent protection, "but we're very pleased with a method that efficiently delivers raw sample," she said. "We're looking at blood, genital swabs, buccal cells; we've done urine; we've done bone marrow. Things like needle biopsies [and] fecal samples … will require a fair bit of off-platform preparation before it can be added."
Acker also pointed out that one of the big advantages of In-Gel as a potential POC platform is its stability. "Part of this [In-Gel] technology is actually preserving all of the reagents and components on the chip for a long time under extreme environmental conditions," he said.
The University of Alberta team has been primarily working under a C$5 million grant provided by Alberta Innovates-Health Solutions, a provincial agency charged with supporting health-related research and technology transfer in the region.
Aquila doesn't directly receive any of this funding, but reaps the benefits of the group's technology development efforts. The company, meantime, has thus far secured about a half million Canadian dollars in private funding, and is currently attempting to raise another C$750,000 over the next two years, Acker said.
"We're projecting it to be about a C$3 million program to get the first test to market," he said. "Once we've done that, hopefully we'll be out of the stage of raising private money."
And, like many early-stage molecular diagnostic companies, Aquila is not averse to partnering with larger, more-established firms in the space.
"We've been in discussions with a number of pharma and biotech companies that are interested, but the challenge is that it's still a little early for them," Acker said. "They're looking for that data from those end users … to reduce their risk, and that's pretty standard, both on the human and veterinary side."
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