Digital PCR was first described in 1992 by PJ Sykes, PhD, et al., and perfected by Bert Vogelstein, MD, and Kenneth W. Kinzler, PhD, in 1999. The technique is a new approach to nucleic acid detection and quantification that offers an alternative to real-time and traditional PCR. Digital PCR offers an accurate and sensitive method that directly counts individual nucleic acid molecules based on on/off signals, providing absolute quantification data.

The sample is prepared in a manner similar to that for real-time PCR but then is separated into thousands of partitions, each containing zero, one or a few template molecules. Partitions with a target molecule will amplify via PCR and a fluorescent probe is used to detect the amplified target (positive reactions).

The ratio of positives to negatives in each sample is the basis of quantification. Digital PCR does not rely on the number of amplification cycles for molecules quantification like real-time PCR does; rather, it relies on Poisson statistical data analysis to determine the absolute template quantity. In the end, the number of positive reactions is proportional to the total number of molecules present in the sample. Because it is not dependent on reference standards, digital PCR data can be compared across instruments or labs, which enhancing collaborations among the scientific community and increases results reliability.

Although it is a relatively young technique and instruments are the priciest among PCR systems, digital PCR is burgeoning in the pharmaceutical and biotechnolgy sector as well as in academic laboratories. It is ideal for applications such as rare mutation detection, pathogen detection and quantification, GMOs detection, NGS libraries quantification and data confirmation, developments for real-time PCR references and others.

Undoubtedly, Bio-Rad Laboratories is the leading vendor in the digital PCR market. Bio-Rad’s QX200, introduced in 2013, is a high-throughput instrument that offers the highest sensitivity and accuracy in terms of absolute quantification among PCR instruments that are currently on the market. RainDance Technologies is the second largest supplier in the niche market of non-diagnostic digital PCR instruments, with its picodroplet technology–based RainDrop ddPCR platform. The RainDrop System is designed exclusively for research use, and is the only unified platform for NGS amplicon enrichment and digital PCR. Thermo Fisher Scientific offers the QuantStudio 3D digital PCR system, which features a chip-based workflow. Other vendors include Fluidigm, Formulatrix and Stilla Technologies.

In 2015, the total market for digital PCR was about $64 million. It has an expected growth rate of 9.0% for 2017, explained by its absolute quantification prowess and utility with NGS applications, as well as its rising number of utilizations in the liquid biopsy arena.

Digital PCR at a glance

Leading vendors:

• Bio-Rad Laboratories
• RainDrop Technologies
• Thermo Fisher Scientific

Largest Markets:

• Biotechnology
• Pharmaceutical
• Academia

Instrument Cost:

• $30,000­–$150,000