Introduction to Digital PCR

Digital PCR (dPCR) is the third-generation PCR technology developed after conventional PCR and real-time quantitative PCR (qPCR). Its core principle is to partition a reaction mixture containing target nucleic acid molecules into tens of thousands of independent micro-reaction units (such as micro-droplets or microwells), with individual PCR amplification occurring within each unit.

After amplification is complete, an endpoint detection (typically of a fluorescence signal) is performed on each unit. The presence or absence of a signal determines whether that unit contains the target nucleic acid molecule. By counting the number of positive units and applying Poisson distribution statistics, the absolute copy number of the target nucleic acid in the original sample can be directly calculated.

The core advantage of Digital PCR lies in its capability for absolute quantification without reliance on a standard curve, leading to more precise and reliable results. Additionally, it has a higher tolerance to inhibitors and demonstrates excellent sensitivity in detecting targets of extremely low abundance, such as rare mutations, trace pathogens, and circulating tumor DNA (ctDNA).

These features have enabled dPCR to show tremendous application value and potential in life science research and clinical diagnostics, including fields such as tumor liquid biopsy, non-invasive prenatal diagnosis, precise pathogen detection, gene expression analysis, quantification of transgenic components, and the valuation of reference standards.

Types of Digital PCR

Digital PCR can be broadly classified into three main types based on the partitioning method used to separate the sample into many individual reaction units:

Droplet-based digital PCR

This method partitions the PCR mixture into tens of thousands tiny water-in-oil droplets, each acting as an independent PCR microreactor. Droplets are generated using microfluidics and then subjected to PCR amplification. After amplification, droplets are analyzed individually for fluorescence to determine positive or negative partitions. This approach offers a very high number of partitions and is widely used for its sensitivity and precision.

Chip-based digital PCR

In this approach, the PCR mixture is partitioned into tens of thousands of microchambers or wells on a microfluidic chip or plate. Each well contains a small volume acting as an isolated PCR reaction. After amplification, fluorescence imaging or scanning reads each partition’s signal. Chip-based dPCR systems often integrate sample partitioning, amplification, and detection in a single device.

Hybrid methods

These include technologies of droplet-based reaction unit and chip-based scan method that combine features of droplet and chip-based systems. Rainsure used this method. First, tens of thousands of droplets are generated using microfluidics, then the droplets are spread in a single layer on a chip. After the PCR reaction is completed, fluorescence signals are collected by imaging in the same way as the chip-based method.

Each partitioning strategy isolates nucleic acid molecules into separate reaction compartments, enabling the use of Poisson statistics to accurately quantify target DNA or RNA molecules by counting positive and negative partitions. The choice of partitioning method affects the number of partitions, partition volume, workflow complexity, and instrument requirements.

Poisson statistics in digital PCR

The Poisson distribution is fundamental to digital PCR because it models the probability that any given partition contains zero, one, or multiple target molecules. By considering these probabilities, Poisson statistics enable the accurate estimation of the average number of target molecules per partition. Specifically, when the average volume of each partition (VP) is known, the Poisson model can be applied to calculate the absolute concentration of target nucleic acid molecules per unit volume.

The key insight from the Poisson distribution is that the fraction of negative partitions (those without any target molecules) reflects the likelihood of zero molecules in a partition, which allows for the calculation of the average occupancy (λ) across all partitions. This approach corrects for the possibility that some positive partitions contain more than one molecule, preventing underestimation of the target concentration.

Accurate measurement of each variable—positive partitions, total partitions, and partition volume—is essential, as any error can directly affect the precision of the nucleic acid concentration determination. Overall, the Poisson distribution provides the statistical framework that transforms the binary positive/negative partition data into an absolute and highly precise quantification of target molecules in digital PCR.

How to calculate copies/droplet and copies/µL in digital PCR?

The total number of copies of the target molecule in all valid droplets of a sample is calculated by multiplying the copies of the target molecule per droplets with the number of valid droplets. Based on the known number of copies of the target molecule per droplet (λ) and the droplet volume, the copies per microliter can also be calculated.

The target DNA concentration in the original 2 mL blood is approximately 35,846 copies/mL.

What is the workflow of digital PCR DropDX series?

The digital PCR workflow is simple. It typically encompasses the following stages:

1. Reaction mix preparation

The digital PCR (dPCR) detection workflow begins with nucleic acid extraction from the sample to isolate DNA or RNA. After extraction, the purified nucleic acids are mixed with PCR mastermix, including primers, fluorescent probes, nucleotides, enzymes, and a specialized supermix designed for droplet formation.

2. Sample loading

3. Droplet generation and PCR

4. Chip reading and data analysis

What is the workflow of digital PCR RS32 series?

The all-in-one digital PCR system RS32 offers a fully automated "sample-in, result-out" workflow. After simple sample preparation and loading the sample onto the chip, users only need to set the required parameters. Once the chip is placed into the RS32 machine, the entire detection process proceeds automatically without any further manual intervention. This streamlined process eliminates the need for hands-on steps during analysis, achieving complete automation from sample input to result output.

Advantages and limitations of digital PCR

Advantages:

Advantage	Description
Absolute quantification	Directly counts target molecules without the need for a standard curve or reference, improving reliability.
High precision and reproducibility	Provides more precise and reproducible results, especially for low-abundance targets and small fold changes.
Superior sensitivity	Detects extremely low concentrations of targets, such as rare mutations, trace pathogens, and ctDNA.
High tolerance to inhibitors	Partitioning reduces the impact of PCR inhibitors, allowing robust quantification even in challenging samples.
No reliance on amplification efficiency	Results are less affected by variations in PCR efficiency, leading to more accurate quantification.

Disadvantages:

Disadvantage	Description
Narrower dynamic range	The limited number of partitions restricts the range of target concentrations that can be accurately quantified, often requiring sample dilution for highly abundant targets.
Higher cost	Equipment and consumables for dPCR are generally more expensive than those for qPCR.
Lower throughput	dPCR typically processes fewer samples per run.
Limited reagent kit variety	Commercialized reagent kits are still limited in variety, and even fewer kits have obtained qualification for use in hospitals.
Non-interchangeable reagent kits	Because the partitioning methods differ, reagent kits from different digital PCR manufacturers are not interchangeable.

Comparison Between Digital PCR and qPCR

qPCR is ideal for high-throughput experiments with a broad dynamic range and fast results. It is quantitative but typically requires standard curves or references, and its accuracy can be affected by inhibitors and PCR efficiency.

dPCR offers absolute quantification without standards, excels in sensitivity and precision for low-abundance or rare targets, and is more tolerant to inhibitors. The choice between qPCR and dPCR depends on the experiment's goals, sample type, and required sensitivity.

Comparative Table: qPCR vs. dPCR

Feature/Aspect	Real-time PCR (qPCR)	Digital PCR (dPCR)
Quantification	Relative or absolute (requires standard curves or references)	Absolute (no standards or references required)
Reaction Format	Bulk PCR, flexible reaction volumes	Sample partitioning, fixed partition volumes
PCR Efficiency Impact	Impacted by changes in PCR efficiency (data collected during exponential phase)	Unaffected by amplification efficiency changes
Tolerance to Inhibitors	Prone to inhibitors	Higher inhibitor tolerance
Detection Method	Real-time detection	End-point detection
Dynamic Range	Broad dynamic range	Narrower dynamic range
Sensitivity	Higher variation, less sensitive to rare targets	Detects small changes and rare targets, higher sensitivity
Reproducibility	Moderate, can be affected by various factors	Higher reproducibility
Statistical Power	Lower	Higher
Protocol Maturity	Well established protocols	Easy transition from qPCR, but protocols still developing
Throughput	High	Lower

Comparison Between Digital PCR (dPCR) and Next-Generation Sequencing (NGS)

Digital PCR (dPCR) and Next-Generation Sequencing (NGS) are complementary technologies in molecular biology and clinical diagnostics. Rather than competing, they are often used together to maximize analytical power:

NGS is ideal for broad discovery, identifying a wide range of genetic variants and biomarkers without prior knowledge of mutations. It is fundamental for comprehensive dPCR provides ultrasensitive validation and precise quantification of specific targets, making it suitable for tracking known mutations or rare variants over time, especially in monitoring treatment response.

NGS	Digital PCR	Digital PCR
Detection Limit	10%-5% for WGS and WES	0.1%-0.001%
Data Analysis	Extensive bioinformatics support, demanding data interpretation	Straightforward
Mutation Knowledge Requirement	Prior knowledge of mutations not required	Required
Scope of Detection	Hundreds to whole exome or whole genome	Limited
Quantification	Semi-quantitative	Absolute quantification
Types of Alterations Detected	Copy number alterations, structural rearrangements, SNV or methylation changes over whole genome/panels	Possible on single genes/regions
Turnaround Time (TAT)	Long	Short
Cost	High	Low

These technologies are often integrated in workflows: NGS for discovery and profiling, followed by dPCR for sensitive, quantitative monitoring of selected targets. This synergy is particularly valuable in precision medicine, oncology, infectious disease monitoring, and genetic testing.

Applications of Digital PCR

Citations

1.Mu H, Zou J, Zhang H. (2024). Quantitative Detection of T315I Mutations of BCR::ABL1 using Digital Droplet Polymerase Chain Reaction. Hematol Transfus Cell Ther. 17:S2531-1379(24)00030-0. doi: 10.1016/j.htct.2023.12.007.

2.Zhao Z, Wang Y, Kang Y, et al. (2024). A Retrospective Study of the Detection of Sepsis Pathogens Comparing Blood Culture and Culture-independent Digital PCR. Heliyon. 10(6):e27523. doi: 10.1016/j.heliyon.2024.e27523.

3.Mao S, Lin Y, Qin X, et al. (2024). Droplet Digital PCR: An Effective Method for Monitoring and Prognostic Evaluation of Minimal Residual Disease in JMML. Br J Haematol. 204(6):2332-2341. doi: 10.1111/bjh.19465.

4.Chen J, Liu X, Zhang Z, et al. (2024). Early Diagnostic Markers for Esophageal Squamous Cell Carcinoma: Copy Number Alteration Gene Identification and cfDNA Detection. Lab Invest. 104(10):102127. doi: 10.1016/j.labinv.2024.102127.

5.He Y, Dong L, Yan W, et al. (2024). A Multiplex PCR System for the Detection and Quantification of Four Genetically Modified Soybean Events. Resear Square. doi: 10.21203/rs.3.rs-4766822/v1.

6.Dong L, Li W, Xu Q, et al. (2023). A Rapid Multiplex Assay of Human Malaria Parasites by Digital PCR. Clin Chim Acta. 539:70-78. doi: 10.1016/j.cca.2022.12.001.

7.Kopylova KV, Kasparov EW, Marchenko IV, et al. (2023). Digital PCR as a Highly Sensitive Diagnostic Tool: a Review . Mol Bio (Mosk). 57(5):771-781. doi: 10.31857/S0026898423050051. [Article in Russian]

8.Lu R, Wang J, Li M, et al. (2022). Retrospective Quantitative Detection of SARS-CoV-2 by Digital PCR Showing High Accuracy for Low Viral Load Specimens. J Infect Dev Ctries. 16(1), 10-15. doi: 10.3855/jidc.15315.

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