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UVA Researchers with ZyGem Ties Develop Multiplexed Chip-Based Warfarin Genotyping Assay


By Ben Butkus

University of Virginia researchers led by James Landers have developed a warfarin-sensitivity genotyping assay that combines multiplex PCR and microfluidic electrophoresis on a single chip and demonstrates 100 percent concordance with validated genotyping methods.

The scientists are now validating the assay with Landers' microfluidic, chip-based, infrared PCR method to create an integrated warfarin-response test that can provide results in approximately one hour, Landers, a professor at UVA and CSO of biotech firm ZyGem, told PCR Insider this week.

In addition, the UVA team is working with ZyGem to add the company's proprietary sample prep technology to the mix and port the entire workflow to ZyGem's benchtop sample-to-answer testing platform for further clinical validation, according to ZyGem CEO Paul Kinnon.

ZyGem is in the nearer term collaborating with Lockheed Martin to develop its DNA testing platform for applied markets such as forensics and biodefense (PCR Insider, 9/23/2010). However, when ZyGem acquired the technology along with Landers' startup MicroLab Diagnostics in 2010, it noted its ambition to eventually move into the in vitro diagnostics space (PCR Insider, 5/25/2010).

"ZyGem's vision for the MicroLab platform … has always been to develop the applied market technology first, make a robust product, then take it to a 510(k) filing and do clinical assays," Kinnon told PCR Insider this week. "We've developed a wealth of technology and assays that are applicable to [nucleic acid] extraction, PCR, and electrophoresis."

"We'd love to have [the warfarin sensitivity] assay readily available on our platform," Kinnon added. "The goal is to work with [the Landers lab] to help facilitate that … and later this year start doing those tests on the pre-production units we're currently building for the applied markets, and then find strategic and clinical partners to help us move those products through the pipeline like we're doing with Lockheed."

ZyGem is on schedule to have its pre-production MicroLab units — called RapID under the Lockheed Martin collaboration — available for forensics applications in mid-2012, Kinnon said.

Landers and colleagues' assay development work, described in a paper published online this week in Clinical Chemistry, is a "first step" toward eventually developing a fully automated assay for ZyGem's platform that physicians could perform at the bedside in an hour or less, Landers said.

"What attracted us to this were the stats on the number of emergency room visits that were related to warfarin dosing," Landers said. "If you take a look at the literature, the mutations that are responsible for causing those differences in metabolism from patient to patient are very well defined."

While there are a number of lab-developed and commercial assays available for warfarin sensitivity genotyping, the turnaround time for these tests ranges from days to weeks — far too lengthy for the acute setting, where physicians must make dosing decisions within a few hours. A point-of-care test that can provide results in under an hour would address this drawback.

"If you take the next step and look at the instruments that are out there for this — and there are a whole slew of them — it became clear that a microfluidic platform had the opportunity to bring something to the table. So this paper really is just the tip of the iceberg," Landers said.

As part of their initial study, Landers and graduate student Brian Poe designed and optimized primers for a fully multiplexed assay to examine three bi-allelic SNPs affecting warfarin sensitivity: CYP2C9 *2, CYP2C9 *3, and the VKORC1 A/B haplotype.

In searching the literature for a method that would enable a single PCR amplification followed by microfluidic electrophoretic separation, Poe identified the amplification refractory mutation system, or ARMS, and a multiplexed version of the assay called tetraprimer ARMS, or T-ARMS.

Both methods had been previously applied to pharmacogenomic testing, but a fully multiplexed T-ARMS assay for warfarin genotyping had not been described, according to the researchers.

"The first assay design used ARMS with two different primers going in the same direction," Poe said. "All three SNPs are bi-allelic, so it would be nice to know both the mutant and the wild type at the same time," which a T-ARMS assay using three primer pairs enabled, he added.

The UVA researchers first tested their assay using conventional PCR performed on a Bio-Rad MyCycler thermal cycler, followed by microfluidic electrophoretic analysis on an Agilent 2100 Bioanalyzer.

They analyzed 35 human genomic DNA samples, several of which had genotypes conferring warfarin sensitivity with both homozygous and heterozygous genotypes for each of the three SNPs. Then, they compared the results of their assay with other validated methods such as an Invader assay, allele-specific PCR, and bidirectional sequencing of PCR products, and found 100 percent concordance.

The researchers developed their assay for use with conventional laboratory technologies because "it was important for us to have something where a clinical chemist could access the material and instrumentation, [and] then do the assay," Landers said. Turnaround time for this assay was approximately 75 minutes, which included about 45 minutes of thermal cycling time.

However, as described in their Clinical Chemistry paper, the researchers also conducted proof-of-principle experiments on a dual-microchip platform capable of performing both infrared PCR and electrophoretic separation in order to improve speed and reduce reagent consumption.

The combined version of the assay was able to correctly profile two of the samples with a turnaround time of about 60 minutes per test. Landers said that since submitting their paper to Clinical Chemistry, the researchers have validated the dual-microchip method on additional samples.

"The amplification on the chip is to just get people ready for what's coming down the pipe," Landers said. "That is, if you can do electrophoresis … and PCR … both on chips, in this case different chips, then the next logical step is to interface those on the same device."

"Then, you'd have to have upstream liberation of DNA that is PCR-ready — enter ZyGem," Landers added. "It's relatively easy to think about a simplistic device that would allow you to put a cheek swab into a receptacle, put in some of the ZyGem reagent that digests everything except the DNA; move that DNA with some master mix into a PCR chamber; cycle for 15 minutes; take an injection out of there into the separation domain; separate and detect by fluorescence; and kick out an electropherograph. We're not there yet. That's where we're going, and this is the first step."

Landers and Poe said that they have filed a provisional patent around their T-ARMS assay method. The patent application is currently under review, but Landers said that he believes "we've got something unique here with the sequences."

Poe agreed, stating that "the empirically determined sequence is the unique product. Also I've never seen three T-ARMS in one reaction, because primers don't play along very well."

Landers, Poe, and ZyGem are now discussing ways to move their assay to an integrated system like ZyGem's MicroLab platform.

If successful, the assay could be the first in a string of in vitro clinical diagnostic tests for the ZyGem system, particularly those that would benefit from a high degree of multiplexing, Kinnon said.

"Genotyping is a great area for us to go into, and infectious disease is another one," Kinnon said. "We have a patent pending for a multiplexed application called microbial fingerprinting, which can identify [up to] 30 different infections on one assay. So that's one of the assays we'll also bring into play."

"In the next 18 months we'll do proof of principle on these clinical assays, including the assays that [Landers' lab] has developed, and some of the infectious disease assays, and then we'll target specific clinical partners to help move those forward," he added.

Have topics you'd like to see covered in PCR Insider? Contact the editor at bbutkus [at] genomeweb [.] com.