Danish diagnostic technology developer Atonomics has published a paper describing its proprietary nested PCR technology and has demonstrated its ability to simultaneously detect multiple yeast and bacterial nucleic acid targets more quickly, and with a lower limit of detection, than standard nested PCR.
The technology, dubbed IsoPCR, combines a single multiplex preamplification PCR with subsequent detection of specific targets using isothermal amplification, and could provide molecular diagnostic developers with a tool to create disease assays or biomarker panels that are sensitive and highly multiplexed — with perhaps as many as 50 targets.
Having filed patent applications for its method, Atonomics is now weighing its options to either develop a molecular testing instrument platform to perform assays based on the technology, or partner with another company that already offers a suitable system, Peter Warthoe, CSO of Atonomics, told PCR Insider.
Privately held Atonomics was founded around 10 years ago, and is owned by a number of other companies and individuals including Inventages (a Nestle company), Beckman Coulter, Sunstone Capital, NeuroSearch, MuRata Manufacturing, and several Atonomics founders, including Warthoe.
The company has focused most of its resources to this point on an immunodiagnostics partnership with Beckman Coulter. Specifically, when the companies entered into an equity agreement in May 2011, Atonomics had been developing the Atolyzer, a desktop, touchscreen instrument designed to run a point-of-care version of Beckman's Troponin I assay for aiding in the diagnosis of heart attack. Last month, Atonomics achieved a milestone in this agreement, triggering an undisclosed payment from Beckman.
"The milestone that we hit [is that] we now have the same performance as a [larger] instrument," Warthoe said. "That doesn't mean we have an instrument ready for sale, but now we can execute an assay with the same performance characteristics as a central laboratory."
In the meantime, however, Atonomics has also been developing a molecular testing technology platform — specifically, the IsoPCR technique.
In so-called nested PCR, which is the technique employed by many well-known molecular diagnostic vendors, including Cepheid, target DNA undergoes a first PCR using primers that yield several unwanted amplification products as well as the target. Then, the amplified target DNA undergoes a second, more specific PCR with different primers, this eliminating unwanted gene products and enhancing sensitivity.
IsoPCR, as described by Atonomics, is a "nested-like" two-step amplification method. In the first step, multiplex PCR is performed on all potential target DNA in a sample using a forward inner primer, or FIP, and backward inner primer, or BIP.
Standard PCR thermocycling subsequently incorporates the 5' end into resulting amplification products. Those amplification products that contain desired sequences from the FIP and BIP primers from the first step are then split into distinct physical chambers, tubes, or zones, and serve as a starting point for a second-stage isothermal amplification that also uses additional loop primers to accelerate the reaction and yield a lower limit of detection for each target.
The upshot of the method is that it offers a high degree of multiplexing without compromising sensitivity, according to the company.
"With IsoPCR we are addressing the fact that it's pretty difficult to have high multiplex[ing] and high sensitivity at the same time," Warthoe said. "For example, if you want to do sepsis detection, it's important [to] detect a lot of different bacteria, at least 35 or 40. But at the same time, it's also extremely important to have high sensitivity."
Warthoe added that several studies claim an effective test for sepsis-causing organisms should be able to detect at a level below 10 colony-forming units per milliliter of blood. "But, if you have 10 CFUs in the beginning and … you spread your PCR reaction from the start in different zones you will lose your sensitivity just [due to the] fact that … with different multiplex reactions … there will be less than one [gene] copy per reaction."
In addition, the use of isothermal amplification for the second amplification step greatly reduces the potential cost of the technique and of the instrumentation necessary to perform it. Warthoe said that most isothermal amplification methods — strand displacement or helicase-dependent amplification, for instance — can be used. However, Warthoe said, "there are a lot of different techs out there for [isothermal amplification] … and I've tried almost all of them. I believe the best isothermal amplification technology out there is LAMP."
But even LAMP, he added, has some of the same drawbacks as PCR, particularly the inability to multiplex while maintaining sensitivity. "What we are doing with IsoPCR is taking the best from PCR and the best from LAMP and combining them," he said. For instance, another benefit of the IsoPCR method is that it actually works better if only a limited number of thermal cycles are run – 10 to 20 as opposed to 30 or more – for the bulk PCR amplification. As such, the technique could be a time saver, as well, cutting assay performance times down by about half.
In a paper published last month in Clinical Chemistry, Warthoe and collaborators at Atonomics described IsoPCR in detail and used the technique to detect DNA from the yeast Candida glabrata, detecting one copy as opposed to a nested qPCR assay that could detect no fewer than five copies. In addition, they determined that the IsoPCR assay had a lower limit of detection than a standard LAMP assay and a nested LAMP assay. Lastly, the researchers demonstrated that IsoPCR could simultaneously detect sepsis-related pathogens with individual limits of detection of 10 copies or fewer.
Warthoe said that the company has also demonstrated how IsoPCR can be modified into a method called RT-IsoPCR to detect specific human gene signatures. They also have used this method, which incorporates a reverse transcription step, to successfully run CardioDx's Corus CAD test, a 24-mRNA assay that can be used to help diagnose coronary artery disease. This research will be the subject of a soon-to-be-published paper, Warthoe said.
"You could make a small IsoPCR chip so you could easily do a 24-RNA profile in about a half hour," he said. "Right now CardioDx's test is running on large instrumentation using real-time PCR, and it costs a lot of money and takes a lot of time. But with our technology you could do RT-IsoPCR, and actually get a result for those genes within a half hour and tell the patient on the spot whether they have this disease or not."
Atonomics has applied for patents on its technology, and its next step is to either build or find an existing instrument platform to execute assays using the technology.
Warthoe said that the assays could run on many existing real-time PCR platforms, but that most existing instruments are more expensive and have more features than are necessary to run IsoPCR tests.
"Definitely we will build the instrument … maybe alone, maybe in partnership," Warthoe said. "We would like to amplify in separate zones, and don't like combining dyes like a Roche [or] Cepheid is doing. Each amplicon should be amplified in a different zone.
"But we don’t believe the instrument is so important. Right now the best on the market, in my opinion, is the [FilmArray] system from BioFire [Diagnostics]. It takes about one hour, and we believe we can do much better than that in the future. With IsoPCR, we can take the time down to about a half hour with the same sensitivity.
In addition, Warthoe added, "instead of paying [about $50,000] for an instrument, we would like to put it a little more in [point-of-care] format. We are not saying it should be $1,000, but it should be a little cheaper [than $50,000]."
The company is looking to take the next step with its technology soon. Atonomics has enough funding for about another year, Warthoe said, and it is still heavily focused on the Beckman Coulter troponin collaboration.
"Right now [we are] funded by our owners, and right now we are discussing what we should do," he added. "It could be that we separate out in a US entity. If we want funding for several years, we need to start looking now. And we are looking into that. Right now we believe we have a technology that could take the next step of multiplexing and sensitivity."