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Roche Developing Digital PCR Platform


This article has been updated from a previous version to include comments from an independent third-party digital PCR expert.

SAN ANTONIO (GenomeWeb) — Roche revealed at the Association for Molecular Pathology annual meeting here yesterday that it has been developing a digital PCR system at its Roche Molecular Systems branch in Pleasanton, California.

Company representatives at the meeting said that Roche plans to commercialize the system in Europe with CE IVD marking for clinical applications and in the US with a 510(k) exemption, though they did not disclose a timeline.

The new system would directly compete with Bio-Rad's QX200 Droplet Digital PCR system, widely regarded as the dominant player in the field.

Digital PCR, and especially droplet-based digital PCR, has gained popularity for molecular biology and clinical applications necessitating detection and absolute quantification of extremely rare mutations or other nucleic acid targets.

The general principle involves taking a sample and compartmentalizing it into individual partitions — generally droplets or microfluidic wells — for direct absolute quantification. When the sample is divided, each compartment will statistically contain either zero or one, and occasionally two, copies of the target. All the compartments also have the necessary reagents for a PCR reaction, which then amplifies the target in partitions that contain it to a detectable level, producing a binary readout.

Generally speaking, the greater the number of partitions, the more accurate the system is, although there are exceptions and tradeoffs. Given its ability to perform absolute quantification, digital PCR has become an ideal platform for applications such as liquid biopsy, viral load monitoring, and copy number variation analysis, among others.

For several years, there were only two main competitors in the droplet-based digital PCR space: Bio-Rad and RainDance Technologies, until Bio-Rad acquired RainDance in early 2017. For plate- or chip-based platforms, Fluidigm was an early player but the architecture of its chips somewhat limited the ability to conduct highly partitioned digital PCR assays, while Thermo Fisher Scientific has more successfully penetrated the market with its relatively inexpensive QuantStudio 3D system. Meanwhile, newer entrants such as French firm Stilla Technologies, Boston-based Formulatrix, and Singapore's JN MedSys are still finding a foothold.

At a corporate workshop session at AMP yesterday, Nick Newton, principal scientist and R&D manager for qPCR systems at Roche, provided an overview of his company's planned entry, which is still in the prototype stage and not yet in the hands of early-access users.

The system will comprise two instruments, a small desktop partitioning engine and a larger benchtop analyzer. It also includes analysis software, which can be run on a local laptop or desktop computer. In terms of its components, the platform is very similar to the Bio-Rad QX200.

Sample will be added manually to consumable plates each having eight sample lanes or channels per plate. Capillary action then draws the sample along the channels where it begins naturally settling into partitions. The plates are then placed into the partitioning engine, which adds a silicon oil sealant that pushes the sample farther down the channel to the end and seals the partitions, a key aspect to being able to perform individual PCR reactions. The partitioning engine will feature touch-screen operation, and partitioning will take approximately 2 minutes per plate, Newton said.

Roche intends to offer three different types of plates depending on the application: a universal plate with 30,000 partitions; a high-sensitivity plate with 20,000 partitions that will maximize sample input volume; and a high-resolution plate with 100,000 partitions but reduced volume tradeoff. In comparison, the Bio-Rad platform features one plate that divides a 20-µl sample into 20,000 partitions.

The Roche system will use a generic DNA/RNA master mix at 5X concentration and a filling dye control, used to invalidate any partitions that are not filled correctly.

The Roche digital PCR analyzer will feature fully integrated thermal cycling and partition imaging of the sealed nanowell plate, with a 12-plate capacity and 96-sample batch size. Perhaps the system's most differentiating feature will be its six-channel detection, which allows for higher target multiplexing than any digital PCR platform — Bio-Rad's system, for instance, has two detection channels.

Roche's new system may be represent another step forward in digital PCR innovation, according to Mikael Kubista, chairman and founder of TATAA Biocenter, a Europe-based provider of qPCR and NGS research services and educational programs.

"Compared to the present platforms on the market and the Bio-Rad ddPCR [platform] in particular, the option to analyze larger total volume is welcome, as it will increase sensitivity," Kubista said in an email. "The alternative extreme option to compartmentalize the sample into 100,000 partitions is particularly attractive when measuring rare mutations and when multiplexing with strategies that require clonal amplification."

Kubista, who is also head of the department of gene expression at the institute of Molecular Genetics, Czech Academy of Sciences, also noted that another potential advantage of the Roche platform is that generic reverse transcriptase and master mixes can be used, enabling higher optimization of assays targeting sequence variants.

"Still, at the end of the day, most important in a digital PCR workflow is the ruggedness, comprising not only the analytical readout, but the entire workflow from sample preparation to analysis of the data," Kubista said. "Bio-Rad has been most successful developing a robust workflow offering very high inter-laboratory reproducibility, which is key to reliable diagnostics. It remains to be seen if the Roche system will match the excellent performance of the QX200."

Newton said that Roche has applications in development that will take immediate advantage of this six-target capability, chief among them copy number variation detection. He also noted that amplification or deletion of genomic loci is associated with various genetic diseases, and the multiple detection channels will allow detection of more than one target, plus multiple reference genes.

Newton also briefly discussed the possibility of performing microsatellite instability detection, noting that the company has at least theoretically designed assays for five MSI markers (mononucleotide repeats) that will be able to identify alterations as small as a two-base deletion.

After Newton's presentation, Harsha Mokashi, international product manager for genomics and oncology for Roche, said that Roche plans to commercialize the system in Europe and elsewhere with CE IVD marking. It plans to register the system as an in vitro diagnostics device in the US with 510(k) exemption because it is essentially a general all-purpose thermal cycler device.

Mokashi declined to provide a timeline for commercialization or a potential price for the platform.