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Isothermal Quadruplex Priming Amplification Moves Closer to Point-of-Care MDx Applications

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NEW YORK (GenomeWeb) — In an effort to design primers that might solve the conundrum of the plateau effect in PCR amplification, researchers at Ohio State University and Ilia State University in the country of Georgia have created a method called quadruplex priming amplification.

The plateau-free method also happens to be isothermal, and is intrinsically fluorescent, making it suitable for use in point-of-care diagnostic assays — an application the OSU group is now working with unnamed entities to develop.

The isothermal QPA method was initially funded by the Bill and Melinda Gates Foundation in late 2011, as reported in PCR Insider. There have been a flurry of publications on the method this year, and it is so new that it didn't make it into a February Molecular BioSystems review of isothermal methods or a December 2013 Methods review of quadruplex-based methods for nucleic acid detection.

However, it has numerous advantages over other isothermal techniques, Besik Kankia, one of the method's inventors and a senior research scientist at OSU, said in an interview this week.

The method is designed to be very simple. It uses the free energy of a single primer to drive the amplification reaction. By comparison, loop-mediated isothermal amplification (LAMP) requires six primers, Kankia said. The reaction is also monitored by intrinsic fluorescence of the primer, which is constructed with fluorescent nucleic acids, whereas all other isothermal methods use external detection methods.

The plateau problem, which occurs in late stages of amplification and causes PCR product yields below theoretical maximums, lured Kankia, a DNA biophysicist, into this field. He felt that self-annealing of DNA and overly complex reactions were the root causes of suboptimal PCR yields.

The QPA reaction is based on formation of "an unusually stable monomolecular quadruplex" by a guanine rich sequence constructed with fluorescent nucleotides, Kankia said. These are quenched by neighbor nucleotides until a quadruplex is formed.

Interestingly, unlike other isothermal methods, such as LAMP or helicase-dependent amplification (HDA), which can only amplify very short segments of DNA, the QPA method can amplify hundreds of basepairs, generating "long DNA molecules for downstream applications, like sequencing," Kankia said.

Kankia originally described the QPA primer in a 2011 Analytical Chemistry paper. His group then reported its efforts to optimize intrinsic florescence in the reaction.

A subsequent Biophysical Chemistry paper highlighted that linear QPA could be done at constant temperature.

But so far this year, the group has published five studies on QPA, describing its modification with a new fluorescent nucleotide and compatibility with a range of temperatures and polymerases. The researchers have also shown how they are developing the isothermal reaction combined with nicking enzyme as a diagnostics tool, and evaluated the QPA assay in surrogate patient samples containing mRNA biomarkers.

Finally, earlier this month, a study in Biopolymers revealed they have made the liner method exponential, with 1010-fold amplification in less than 40 minutes.

Ready for prime time?

Isothermal PCR is already being extensively used in diagnostic assays, although most require at least a benchtop heating block and thus are not truly point-of-care handheld devices.

For instance, Meridian Bioscience offers a number of FDA-cleared and CE marked assays based on LAMP and sold under the brand name Illumigene. And earlier this year, Alere launched the US Food and Drug Administration-cleared Alere i and is actively developing tests and pursuing CLIA waiver. It also hopes to launch a quantitative HIV assay on a second platform, called Alere q, in Africa in the first quarter of 2015. These two platforms have the potential to use either isothermal nicking enzyme amplification reaction (NEAR) or recombinase polymerase amplification (RPA). Earlier this month, the company also received a $12.9 million contract to further develop the devices and reduce their costs below the current $8,500 per platform and $80 per test.

Quidel's Gates-funded Savanna and Solana platforms, meanwhile, use the aforementioned HDA. That firm recently reported it is aiming to pull forward the timeline for testing HIV assays on Savanna alpha units in Africa. The company's AmpliVue molecular diagnostic product also uses HDA, which it claims can reduce assay times to 20 minutes.

Kankia's group, meanwhile, has also recently reduced the QPA assay running time to 20 minutes, he said. The method described in Biopolymers used magnetic beads, but the researchers are currently developing it into a single-tube assay.

Now, they are working with engineers to write a grant for development of a platform. Since the reaction only requires a single constant temperature, they believe it will be a handheld, battery-operated device costing about $500, with assays in the $1 range.

For the past three years, the Bill and Melinda Gates Foundation funded this research for the purpose of developing a molecular diagnostic for low-resource settings. However, OSU owns the IP and the group is collaborating with outside entities to develop the assay further, although Kankia was not at liberty to name the parties.

For diagnostic use, "the DNA detection is universal; the PCR could be adjusted to any disease," Kankia noted.

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