By Ben Butkus
PCR amplification of nucleic acids in less than three minutes using typical microliter-scale reaction volumes is now possible, thanks to a team of researchers from Lawrence Livermore National Laboratory.
The dramatic time savings — current non-microfludic thermal cycling methods at best take about 10 minutes — could enable the development of near-instantaneous and highly specific nucleic acid-based diagnostic tools for stemming potential infectious disease pandemics in their earliest stages, the scientists said.
To that end, the group is now modifying its technology to improve the performance of the polymerase enzymes used and to incorporate real-time detection; and is seeking commercial partners to help bring its goal to fruition.
"It's ready for commercialization," Reginald Beer, a principal investigator at LLNL and one of the technology's inventors, told PCR Insider this week. "We have IP that has been filed, and we [are preparing] additional applications. There is IP that is available for licensing."
Beer's background includes a stint in the laboratory of former LLNL researcher Bill Colston, who went on to found QuantaLife and who helped develop some of the technology used by both QuantaLife and RainDance Technologies in their emulsion-based digital PCR offerings.
Though Beer believes that digital PCR is a promising technology for many applications, and is suited for ultra-fast amplification in individual microdroplets, he shifted his interest toward pushing the boundaries of the amount of time needed to conduct standard PCR amplification in what he called "volumes that are real."
"It's really great and sexy to do things on the picoliter scale," Beer said. "But when people actually use these systems … invariably someone says, 'OK, I've made my emulsion, I've run my reaction, now I want it back. I want to sequence it, or do something with it. And they're left with how to get their material out of a 10-picoliter droplet. How do you manipulate and interface with that?"
Instead, Beer wondered, "what if we could make PCR so fast that we could start bringing it into the diagnostic realm, the point-of-care realm? If you can bring the specificity and sensitivity of PCR, which can't be beaten, to molecular diagnostics that can be performed in the ten-minute or five-minute range, it could have a huge impact."
A research study published by Beer and colleagues last week in the journal Analyst represents "the first step down that path," Beer said.
In their paper, the researchers briefly describe the prototype device as "achieving its accelerated thermal ramp rates from superior heat transfer due to convective flow through a porous media substrate, which is augmented by a thin-film resistive heater."
The system, which used 5-µL reaction volumes, has maximum heating and cooling rates of 45 °C per second, allowing extremely short thermal cycles; and the time constraints associated with the heating and cooling ramps of a single cycle are approximately 1.02 and 1.32 seconds, respectively, according to the paper.
Perhaps a bigger challenge than designing hardware for sub-three-minute PCR was identifying a polymerase that could work at the speeds envisioned by Beer and colleagues. Beer said that the group identified 10 commercial polymerases that were advertised as working at the required speed, much faster than the commonly used Taq polymerase.
However, only two of these enzymes worked in the researchers' device right out of the box: SpeedStar HS DNA polymerase from Takara Bio and KAPA2G Fast PCR enzyme from Kapa Biosytems.
"We started with a much larger group of enzymes, and we just didn't get results," Beer said. "We tried some that we thought should have worked but didn't on our system. The reason we didn't publish those results in the paper is that I think it is unfair to the enzyme manufacturers, because none of them were given time to adjust assay components or anything like that. So I don't want to say that enzyme X wouldn't work."
Even the Takara and Kapa products, while adequate, could be tweaked for faster performance. "There are a lot of parameters you can adjust," Beer said. "We adjusted magnesium chloride on some; we didn't really get any benefits from that. We did seem to get better performance out of adding more polymerase, so that's another thing we can think about."
Using their device, the researchers attempted to amplify two different starting templates: genomic DNA from an gram-negative bacterium from the Enterobacter family; or a synthetic oligomer of a fragment of SARS DNA producing three different-length amplicons of 58, 107, or 160 base pairs. They chose the former to demonstrate extreme cycling rates on large genomic DNA; and the latter due to its importance as a public health threat "and exemplar of the potential benefits of ultra-fast PCR in rapid screening," according to the paper.
Their device enabled 30-cycle PCR amplification of all template DNA in as little as two minutes and 18 seconds. According to the researchers, the fastest laboratory-scale-volume PCR with a comparable number of cycles to date took about 10 minutes; although some microfluidics-based techniques in much smaller volumes have broken the 10-minute barrier.
"We previously published a numerical simulation that told us this could actually work," Beer said. "When I first set out to do this, people said it would never work. They'd give me mechanical reasons; or enzyme kinetic reasons; and my answer was always: Well, nobody really knows because we've never had something that can push the enzymes that fast."
In addition to advancing the understanding of the types of fast polymerase enzymes used in their research, the new technology "holds the promise of providing near-instantaneous and specific nucleic-acid-based diagnostics, which will be critically important during future public health emergencies," the researchers wrote.
Besides further optimizing the performance of the fast polymerase enzymes, Beer and colleagues have turned their attention to developing a real-time version of their PCR device.
"We already have a prototype in house that we're working on for [real-time detection]," Beer said. "We haven't coupled it yet to the instrument itself, so we don't have results. But that's going to be the next publication. That's definitely on our path, but we just wanted to nail the instrument first before putting the optics on it."
Down the road, the researchers are envisioning a sample-to-answer PCR-based testing platform that can complete a test in five to 10 minutes.
"We want the sensitivity and specificity of true molecular diagnostics," Beer said. "We want to integrate simple sample collection methods like finger sticks and swabs; to streamline instrument operation and sample handling for CLIA waiver; and ultimately we want to be able to network the results, if [the device] is in the field, perhaps to a remote lab."
Ideally this would all be achieved with the assistance of a commercial partner: "We are in that space at LLNL where we can invent a technology, but we're not in the [business] of taking it across that gap and turning it into a product," Beer said.
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