The new absolute in PCR – Digital PCR

In one sentence, digital PCR is used to detect rare sequences, copy number variations and microbial targets; to quantify of rare mutations, including from liquid biopsies; for single-cell analysis; measurement of viral loads; to analyze gene expression; and to quantify next-generation sequencing (NGS) libraries and many others.

The difference between PCR and digital PCR is that end-point, or conventional PCR, offers qualitative or semi-quantitative analysis, whereas dPCR offers absolute quantification. Digital PCR is faster, more precise and reproducible, it offers multiplexing capabilities, but it also requires a larger initial investment.

The benefits of digital PCR include absolute target quantification without standards or reference curves, high tolerance to PCR inhibitors, high precision needed to detect small-fold changes, high sensitivity, low limit of detection and high reproducibility, especially across different laboratories.

The disadvantages of digital PCR include a slightly narrower dynamic range than qPCR. The method is not ideally suited to large amplicons.

The QIAcuity instrument software does not allow to read and process a plate without seal. If you would like to perform a dry run please use sealed plate and set up this plate in the QIAcuity Software Suite.

The accuracy of digital PCR varies across dPCR systems and applications. A comparison study of the precision and sensitivity of dPCR vs qPCR showed dPCR lowers the standard deviation of replicates down to three-fold. In terms of sensitivity in mutation detection, qPCR loses reliable quantification at a 10% mutation rate and dPCR at a 0.1% mutation rate.

It’s called digital PCR because it gives you obtain binary on/off or yes/no results. Digital assays can only distinguish between two signals rather than a range of possibilities. So things are either black or white, not 50 shades of grey. For digital PCR in particular, the sensor distinguishes between a positive and negative partition and delivers a yes or no answer on whether your target is present in the partition.

Our QIAcuity digital PCR systems have been developed on a nanoplate-based technology, offering significant benefits over droplet digital PCR (ddPCR) technology. Fixed partitions integrated into the dPCR nanoplate prevent variation in size and coalescence, as seen in the ddPCR method. Besides, simultaneous reading of all partitions of the sample in nanoplates results in a faster readout. Nanoplates are not only user-friendly and easy to pipette just like qPCR but are also amenable to front-end automation. Most importantly, correctly sealed nanoplates prevent sample evaporation and well-to-well contamination. Partitioning, thermocycling and imaging are integrated into a fully automated instrument, delivering results in under two hours vs. more than four hours required by some other digital PCR systems. Moreover, scalable configurations and higher multiplexing capability make QIAcuity digital PCR easy to adapt to all kinds of labs and application requirements. Using the QIAcuity Software Suite, digital PCR experiments, samples and reaction mixes can be defined, assigned to nanoplates, and transferred to the QIAcuity instrument. After the run, data can be analyzed, reports can be created, and data can be exported for remote analysis.

QIAGEN dPCR Assays (such as dPCR LNA Mutation Assays) can be found on https://geneglobe.qiagen.com.

Nanoplate dPCR has the unique ability to precisely measure up to five targets in the same reaction. Find more information here. To learn how to design and develop your own multiplex dPCR assay, check out our site on dPCR multiplexing.

The fluorescent signal in the reference channel is measured to determine the number of valid partitions in a well. In addition, differences in the signal intensities between partitions are normalized and the fluorescent signals in the target channels are corrected accordingly.