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University of Alberta Team Develops Improved Cassette Version of 'In-Gel' PCR Array Technology


A team led by researchers from the University of Alberta and spinout Aquila Diagnostic Systems has published a paper demonstrating the latest advances in its gel array-based PCR technology.

Specifically, the scientists have developed a simple, disposable, and inexpensive cassette combining its previously described hydrogel PCR arrays with new "packaging" technology that enables easier delivery of raw sample, creating a platform that is more amenable to point-of-care diagnostic use and mass production.

They also described the ability of their improved technology to simultaneously detect four sexually transmitted infections from genital swabs and urine, demonstrating its potential usefulness as an "on-the-spot" testing device in STI clinics in both the developed and developing world.

PCR Insider first reported on the In-Gel technology in September 2010, after the University of Alberta group led by researcher 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).

And in a paper published last April in Lab on a Chip, Pilarski and colleagues demonstrated the ability of their technology to simultaneously detect DNA from herpes simplex viruses 1 & 2 together with integrated positive and negative controls from unprocessed genital swabs, achieving 91 percent concordance with conventional PCR (PCR Insider, 4/5/2012).

The In-Gel technology used in these studies featured arrays comprising a multitude of 0.67- to 0.86-µL cylindrical or conical hydrogels, or gel posts. And although the technology performed well in the lab, the researchers conceded that it would be difficult to manufacture and that it needed to be in a fully-enclosed format to be amenable to clinical use.

The group's latest iteration of the technology, described in a paper published earlier this month in Lab on a Chip, integrates the In-Gel technology into a self-contained, disposable gel capillary cassette that avoids the need for pumps or valves in order to deliver raw sample of conduct PCR amplification.

The cassette contains capillary reaction units that can be stored at room temperature for up to three months. The device also simultaneously tests up to 16 patients for two or more targets; accommodates different sample types on the same cassette; contains positive and negative controls; and can be configured in different geometries.

Pilarski told PCR Insider in an email this week that the latest version of the technology, which features innovations developed by team member and lead author Dammika Manage, makes "great strides forward on making point of care accessible and clinically useful."

Pilarski said that the new packaging strategy for the hydrogel technology promotes "efficient sample delivery, something that was difficult with the hydrogel technology when used in the gel post geometry we previously reported."

Described briefly, the new cartridge has individual reaction units consisting of hydrogel reaction mix — everything but template — in a capillary, Pilarski said. The hydrogel mixture is polymerized, and then desiccated, creating a channel through which raw sample can flow. The cassette contains multiple reaction units each with a different primer set, and accepts multiple samples each into individual trenches, each of which is filled with multiple individual reaction units.

Each sample, including different types of sample, can be delivered via micropipette to its own trench of assembled capillary reaction units, Pilarski said, adding that PCR is simultaneous for all the reaction units, including a set of negative controls and positive controls.

"It is very powerful technology and importantly is manufacturable and low-cost, being made with off-the-shelf components — no fabrication costs, no pumps or valves, no complex machining," she said.

In their paper, Pilarski, Manage, and colleagues used their new prototype to simultaneously detect HSV I & II from raw genital swabs; and Ureaplasma urealyticum and Mycoplasma homonis from raw urine in as little as 50 minutes.

The researchers also carried out their tests in a prototype portable instrument for PCR thermal cycling with fluorescence detection of amplified products by melt curve analysis.

According to Pilarski, each reaction unit, including reagents, costs about $0.12. As an example, testing 10 samples for 10 targets on the same cassette, along with quality controls, would cost under $2.00 per sample.

This, she said, "makes possible rapid screening of multiple patients for multiple infections, for example in a sexually transmitted disease clinic, while patients are in the clinic." In addition, she added, the cassettes are "manufacturable and amenable to mass production."

This latter point may be the biggest hurdle yet for the research team. The original In-Gel technology was licensed by the University of Alberta to Aquila Diagnostic Systems, a university spinout that was planning to integrate the technology into diagnostic devices for blood-borne diseases; and for point-of-need testing devices to diagnose disease in livestock.

Representatives from Aquila could not be immediately reached for comment. However, last year Chief Technology Officer Jason Acker said that the company was investigating a number of other applications, with a primary focus on veterinary applications, malaria testing in the developing world, and pharmacogenomic testing.

According to Pilarski, the new technology developed by her group is covered by recently filed patent applications. However, it has not yet been licensed to an external partner.