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At First Digital PCR Conference, Enthusiasm Tempered by Realistic Expectations

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Digital PCR has the potential to supplant quantitative real-time PCR as the assay technology of choice for certain applications, particularly the detection and absolute quantification of low-level DNA targets in oncology and HIV research, industry stakeholders and early users said at a digital PCR conference this week.

However, more education on digital PCR is needed as the technology may not be appropriate for a number of assays for which qPCR is currently the gold standard, particularly situations where simple detection — and not necessarily absolute quantification — of a DNA target is desired, according to several conference participants.

In addition, the overall cost of digital PCR may need to decrease while throughput increases in order for it to be considered a serious contender to dethrone qPCR for certain applications, according to some conference-goers.

Representatives from digital PCR vendors including Bio-Rad, RainDance Technologies, Life Technologies, and Fluidigm gathered in San Diego this week along with technology users from academia and industry to discuss these and other topics at Cambridge Healthtech Institute's Digital PCR Applications and Advances conference, the first scientific meeting dedicated solely to the emerging technology.

The fact that digital PCR now has a dedicated conference alone is a sign of rapidly growing interest in the technology, according to Reginald Beer, medical diagnostics initiative leader for the Center for Micro and Nanotechnologies at Lawrence Livermore National Laboratory, and one of the inventors of droplet-based PCR.

"I see great growth in the area," Beer told PCR Insider in an interview following the conference. "When I was doing my research in 2007, I was told this wasn't going to work … and we did it. It was just such a different world from now, where you go to a conference and there are multiple vendors. It's good to see how it's grown."

Beer, whose presentation at the CHI conference provided an overview of the technology and its recent adoption, noted during his talk that qPCR is a mature technology with a "huge market share," and, as such, proponents of that technology are rightfully skeptical when told that digital PCR is likely to replace it as an everyday laboratory workhorse.

According to Beer, either droplet-based digital PCR — such as the QX100 Droplet Digital system from Bio-Rad or the RainDrop platform from RainDance — or plate-based digital PCR — such as Life Tech's QuantStudio 12K Flex and OpenArray consumables and Fluidigm's BioMark HD system — can be potentially "disruptive" to qPCR "only if the cost falls significantly." Until such time, the technology is likely only ideal for specific applications where users need to pick up a small signal from target DNA — for instance, cancer mutations — in an overwhelming background of wild-type DNA.

However, Beer elaborated in his post-conference interview that certain qPCR technologies, such as the molecular beacons being developed by Fred Kramer's group at the University of Medicine and Dentistry of New Jersey, can achieve similar results, and can do so in multiplex fashion and without losing sample to microfluidics, a current bugaboo of digital PCR.

Digital PCR proponents need to "make sure their claims are well-substantiated … and do their homework, because standard qPCR is a pretty well-established technique, and people know how to do it and are getting it done," Beer said.

Continuing the comparison between qPCR and digital PCR, Jim Huggett, science leader for nucleic acids metrology at UK-based lab services and measurement standards organization LGC, noted during a presentation that, besides the frequently cited advantage of absolute quantification that digital PCR provides over qPCR, the former also is more reproducible and more easily lends itself to standardization.

"Interassay variation is a really big problem … [and] calibration and standardization is a nightmare for qPCR assays," Huggett said.

Huggett also noted that the research community needs "reduced cost and increased throughput" from digital PCR vendors, and specifically from the droplet-based vendors, Bio-Rad and RainDance. Currently, platforms from those vendors cost between approximately $85,000 and $100,000, according to company representatives, and the reagents needed to create the emulsion-based droplets and optimize droplet-based amplification are, on the whole, more expensive than qPCR reagents.

"By and large, I think the community is going to yawn until they don't have to pay more to get the advantages of droplets or digitization," Beer said, echoing Huggett's comments.

In his conference presentation, Bruce Gale, an associate professor of mechanical engineering at the University of Utah, described the various microfluidic projects that his group has undertaken to reduce the cost and increase the speed of PCR and digital PCR.

Gale, who recently detailed his lab's development of continuous flow microfluidic PCR and melt-curve analysis technology (PCR Insider, 9/13/2012), also discussed his group's disc-based digital PCR design, which he believes can both lower the cost and increase the throughput of the technique while retaining the core characteristics of absolute quantification and rare event detection.

That technology, developed along with Scott Sundberg, a former University of Utah graduate student and member of Gale's lab, serves as the basis for university spin-out company Espira (PCR Insider, 6/28/2012).

In her presentation on efforts to validate data from digital PCR experiments, Kerry Emslie, bioanalysis group manager for the National Measurement Institute of Australia, described her lab's experience with both a Fluidgim microfluidics-based system and a Bio-Rad droplet-based platform.

Emslie noted that digital PCR generally produces inherently reliable data, without the need for calibration. This is primarily due to the predictable nature of the Poisson distribution statistics, which describe the probability of individual DNA targets compartmentalizing into individual droplet-based reaction volumes when enough reaction volumes are present, as is the case with droplet-based digital PCR.

However, researchers need to carefully consider other factors that are "extrinsic" to digital PCR — such as sample homogeneity and the accuracy of dilution steps — and can affect the reliability of results. In addition, although droplet-based systems are designed to produce relatively uniform droplet volumes, her group found that droplets generated from different wells can vary in volume, which can also produce uncertainty in calculating target concentration.

Commercial Bent

Being that it is still early days for digital PCR technology, purveyors of the technology — particularly vendors of droplet-based systems, Bio-Rad and RainDance — had a strong presence at the conference, with several poster presentations and talks designed to demonstrate the growing breadth of applications for digital PCR and to educate conference-goers on the technology.

Andy Watson, chief marketing officer and head of global sales for RainDance Technologies, and George Karlin-Naumann, senior director of droplet digital PCR assay development at Bio-Rad's Digital Biology Center, served as panelists in a Q&A session on how researchers can implement droplet-based digital PCR in their laboratories.

Watson noted during the session that RainDance "firmly believes this is a technology that will displace many qPCR applications," but added that potential customers "need education," as digital PCR "might not be the right fit for everything."

When asked by an attendee whether digital PCR required any special equipment — besides the instrumentation platforms and a standard thermal cycler — or any assay development training or special chemistries to implement, both company representatives remarked that it was fairly easy to transition from TaqMan-based real-time PCR to digital PCR experiments — with some caveats.

Karlin-Neumann noted that assays that work with real-time PCR using TaqMan chemistry port over to digital PCR "fairly easily," although "some templates have characteristics that are not tractable."

Watson commented that there can be benefits to using "more optimized chemistries" for digital PCR, which is one of the reasons RainDance this week announced an agreement with IDT for digital PCR assay reagent optimization.

"So far most work has been done with off-the-shelf real-time PCR master mixes," Watson said. "However, there are cases … [such as] the EGFR T790M [mutation] that are historically hard to design probes for." Working with IDT, he added, RainDance has been able to double the sensitivity of previous digital PCR assays for this mutation.

In a follow-up email to PCR Insider, Watson elaborated that the EGFR T790M assay "is difficult for design reasons as it has an upstream repeat, a SNP, and family member homologs. Yet it is an important mutation target in cancer. Due to the design difficulty, we had previously observed mutant-to-wildtype detectability considerably worse than 1:10,000 (which we consider our cut-off for performance). We have increased this to 1:11,000 with the new IDT design."

Watson added that despite this improvement, "EGFR T790M is still one of our least impressive sensitivities … [and] LifeTech TaqMan probes for other assays have demonstrated some of our best sensitivities for other assays (better than 1:250,000). We continue to believe that it is important to have chemistry developments that make digital PCR even better; however our own experience also shows that out-of-the box performance of existing assays is also typically very impressive."

At the conference, Karlin-Neumann said that Bio-Rad has had "a similar experience with a number of cancer mutations, particularly hairpin structures," and that those assays "can improve with improved probe design."

Even though the conference primarily showcased the droplet-based digital PCR technologies, a smattering of presentations and posters focused on the utilization of Life Tech's and Fluidigm's microfluidic plate- or chip-based platforms.

In particular, Wendy Winckler, scientific director of the genomics platform at the Broad Institute, which recently opened a single-cell analysis research institute along with Fluidigm, described how her group has been taking single-cell approaches to studying mutations in chronic myelogenous leukemia using Fludigm Dynamic Array technology. In addition, the group is implementing a modified version of Fluidigm's SNPType assays for genotyping, and the company's C1 instrument for single-cell sample prep for downstream gene expression studies.

And Patrizia Paterlini-Bréchot, a professor of cell biology and oncology and director of France's INSERM, described efforts in her laboratory to employ Life Tech's QuantStudio 12K Flex to detect prenatal aneuploidy through genetic analysis of isolated circulating trophoblastic cells. Paterlini-Bréchot underscored the ability of the QuantStudio 12K Flex to combine digital PCR and qPCR analysis on the same system as a major boon for this work.

Commenting on the conference and the comparison of droplet-based systems to plate-based systems such as the QuantStudio 12K Flex, Paul Pickering, head of digital PCR for Life Tech, told PCR Insider that questions remain regarding where digital PCR technology will be applied next and what it will take to move it beyond the early-adopter phase.

"Emulsion PCR has been challenging for the broader research community to get their arms around in the past and it remains to be seen whether it can make the jump this time," Pickering said. "There was healthy debate [at the conference] around the value of reaction numbers; how to handle false positives in rare target applications, where we feel our real-time digital approach excels; and making sure that we as a community don’t promise more than we can deliver … Our experience is that technology needs to be very simple, robust, and reliable to make the jump to widespread adoption, and that appropriately designed chip-based approaches are the best way to achieve this in digital PCR."

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