Digital PCR Background and Applications

The Origins of Digital PCR

Types of dPCR and dPCR Systems

dPCR vs qPCR: When to Use Which

Despite dPCR’s technological advancements, qPCR is often faster, cheaper, and handles more samples at once, so it is still used for high-throughput screening and broad dynamic range applications. The classical 384-well format and lower reagent costs maximize the efficiency of qPCR in those cases. qPCR allows relative quantitation, and multiplex qPCR is a great way to compare the presence and relative quantity of multiple DNA targets at once, and in real time.

However, qPCR's reliance on primer amplification efficiency can create substantial problems: a 10% reduction in efficiency (from 100% to 90%) can shift calculated concentrations by 2-fold or more.⁷ Ct values above 35–40 cycles become increasingly unreliable, as late-stage amplification risks non-specific product formation and primer-dimer artifacts that cannot be distinguished from target amplicons.

dPCR’s binary endpoint detection eliminates these issues. Because each partition is simply positive/negative, results are independent of amplification efficiency. A partition with 1 copy is counted as positive, just as one with 100 copies.

This robustness extends to inhibitor-tolerant quantification of low-abundance targets: where qPCR Ct values shift unpredictably with crude samples (blood, soil, etc.), dPCR maintains accuracy because inhibitors affect all partitions equally rather than altering exponential curves.⁸

dPCR is also much more precise, resolving up to 1.2-fold copy number differences and detecting rare variant allele frequencies down to 0.1 %, a level of accuracy unattainable by qPCR's analysis of standard curve interpolation.⁹

Figure 2: Comparison of PCR, qPCR, dPCR, and ddPCR, their concept, and readout systems.

Major Applications for dPCR

dPCR entered the market after qPCR was already widely established, so many laboratories have qPCR infrastructure but lack dPCR instruments. The higher capital and per-sample cost of dPCR systems makes adoption and use a deliberate choice rather than the default one. The following section goes over applications where dPCR's benefits justify the investment.

Oncology and liquid biopsy: ctDNA and MRD detection

Non-invasive cancer tests have been on the rise in the last decade. Among those are liquid biopsies, where blood or other fluids like urine or tears can be used to check for the presence of cancer signals.¹³

For solid tumors, oncology treatments cause cell death. This causes small amounts of cell-free DNA (cfDNA), including circulating tumor DNA (ctDNA), to be released into the bloodstream. For blood cancer, DNA or RNA from circulating cells is present in the blood.

After surgery or treatment of tumors, NGS is ideal to know which mutations are present in the tumor, but dPCR can be used for minimal residual disease monitoring and to know when treatments can be stopped, as it shows a limit of detection of 0.1%.^{14, 15}

dPCR tests are approved for the monitoring of chronic myeloid leukemia from blood samples, like the Bio-Rad QXDx BCR-ABL %IS Test or the Sniper BCR‑ABL1 (p210) %IS kit. Both measure BCR-ABL1 RNA more accurately than qPCR.¹⁶

Figure 3. Liquid biopsy (blood) for diagnostic purposes.

Copy number variation and cell and gene therapies

Copy number variations (CNVs) are duplications or deletions of DNA segments, which affect around 12% of the human genome.¹⁷ CNV demands precise quantification for cancer diagnostics (HER2 amplification for HER2 status in breast cancer) and gene therapy safety (vector copy number).

Because of the absolute quantification of nucleic acids, dPCR eliminates reference gene instability, a critical flaw in relative quantification methods, like qPCR, array comparative genomic hybridization, and paralogue ratio testing.¹⁸ dPCR can also be used for integrated viral genome monitoring, like in ongoing HIV infections, allowing total quantification of proviruses to determine HIV persistence.¹⁹

In cell and cell and gene therapy manufacturing, dPCR provides better sensitivity than qPCR for AAV vector genome quantification without standard curves, enabling precise potency dosing and CAR-T transduction efficiency measurements.

Pathogen detection and viral load quantification

dPCR can be used to quantify pathogens from low-count fluids with high inhibitor content. An example is absolute viral load quantification from blood and plasma samples. dPCR has proved more effective than qPCR for cytomegalovirus detection in patients after transplantation, and it also enables more precise cross-laboratory monitoring of intervention strategies.²⁰

The same can be said for environmental samples. In a 24-month SARS-CoV-2 wastewater monitoring study, RT-dPCR was able to accurately detect low viral loads in samples where RT-qPCR returned negative or marginal results (Ct 36–40).²¹

Bacterial detection can also be improved with dPCR. A quadruplex ddPCR  assay was used to simultaneously detect four major foodborne bacterial pathogens (Salmonella Typhi, Staphylococcus aureus, Listeria monocytogenes, and Bacillus cereus) with detection limits of 7–9 copies per reaction, showing equivalent accuracy to plate counting. However, ddPCR took five hours while cultures normally take 2-9 days.²²

Overall, dPCR has many potential applications. While reagent costs and initial investment are higher than those required by qPCR or other methods, the added sensitivity and benefits often outweigh the drawbacks.

This article is part of a 3-part series on digital PCR. Continue reading:

• dPCR assay design and experiment planning
• dPCR data analysis and results interpretation
• Inside the Application Digital PCR (dPCR) Hub

Frequently Asked Questions (FAQs)

References