Applications of Digital PCR in Clinical Diagnostics and Therapeutic Monitoring

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ACHAIKI IATRIKI | 2026; 45(1):25–30

Review

Aspasia Papageorgopoulou, Zoi Anastopoulou, Apostolos Vantarakis

Laboratory of Public Health, Epidemiology and Quality of Life, Medical School, University of Patras, 26504, Patras, Greece

Received: 20 Mar 2025; Accepted: 30 Jul 2025

Corresponding author: Apostolos Vantarakis, Tel./Fax: +30 2610 969 875, E-mail: avanta@upatras.gr

Keywords: Digital PCR, molecular applications, clinical diagnostics

 

Abstract

Digital polymerase chain reaction (dPCR) represents a transformative advancement in molecular diagnostics, offering enhanced sensitivity, specificity, and absolute quantification of nucleic acids. This narrative review explores the principles, advantages, and expanding clinical applications of dPCR, particularly in infectious disease diagnostics, oncology, and genetic screening. dPCR has demonstrated superior performance in pathogen detection, liquid biopsy for cancer prognosis, and prenatal genetic testing, surpassing conventional PCR techniques in precision and reproducibility. Furthermore, its resistance to inhibitors and multiplexing capabilities make it a valuable tool in clinical decision-making and personalized medicine. Despite its advantages, challenges such as cost, standardization, and clinical adoption remain barriers to widespread implementation. Continued technological advancements, including automation and high-throughput platforms, are expected to further integrate dPCR into routine clinical practice, ultimately improving patient outcomes.

INTRODUCTION

The increasing complexity of modern healthcare challenges necessitates the development of innovative and robust molecular diagnostic techniques. Molecular analysis has become an indispensable tool in both preventive medicine and disease diagnosis, a fact that was particularly underscored by the COVID-19 pandemic. The need for highly sensitive and specific diagnostic methods has driven the continuous development of novel molecular techniques, as well as the refinement of existing technologies. Among these, polymerase chain reaction (PCR) stands out as the gold standard for pathogen detection due to its unparalleled accuracy in amplifying and identifying nucleic acids. PCR has played a pivotal role in the rapid detection of infectious agents such as SARS-CoV-2, enabling timely clinical decision-making and epidemiological surveillance. As molecular diagnostics continue to evolve, further advancements in PCR and related methodologies are expected to enhance diagnostic precision, efficiency, and accessibility, ultimately shaping the future of personalized and precision medicine.

Described by his inventor, Mullis, as the method that can detect “almost anything in anyone”, it differs quite a bit from its original version with its evolutions taking up more and more space. Its most recent form, digital polymerase chain reaction (dPCR), had emerged as a useful methodology that is gaining more and more applications in prevention, diagnosis and treatment [1].

Basic Principles and Mechanisms

What is digital polymerase chain reaction and why is it considered special? It is a third-generation PCR reaction that enables absolute quantification of nucleic acids through the compartmentalization of the sample and the simultaneous execution of the same reaction in each individual compartment of the sample. Abolishing the semi-quantitative nature of simple PCR, it carries the possibility of direct monitoring of reactions with a time parameter, with greater accuracy overall and at individual stages of reactions, with immediate quantitative and qualitative recording even in complex samples, small quantities and rare genes [2]. Specifically, dPCR functions by dividing a sample into numerous micro-reactions, each containing zero, one, or a few target DNA or RNA molecules. The amplification of the target sequence occurs within these isolated partitions, followed by fluorescence-based detection of positive and negative partitions. The proportion of positive reactions is then used to determine the absolute nucleic acid concentration through Poisson statistical analysis [3]. This partitioning approach eliminates the need for standard curves, improving the accuracy and reproducibility of nucleic acid quantification compared to quantitative PCR (qPCR) [4].

dPCR provides several advantages over traditional PCR-based methods. One of its most significant benefits is absolute quantification, which does not require external reference standards, thereby reducing variability and improving consistency in results [5]. The high sensitivity and precision of dPCR make it particularly useful for detecting low-abundance targets, such as rare mutations in oncology and low viral loads in infectious disease diagnostics [6]. Additionally, dPCR exhibits robust resistance to inhibitors commonly found in biological samples, making it a superior choice for complex matrices such as blood, tissue, and environmental samples [7]. Furthermore, dPCR facilitates multiplexing, allowing the simultaneous detection of multiple genetic targets in a single reaction, which is particularly beneficial in clinical applications requiring comprehensive genetic profiling [8]. However, despite the automation it offers, user training is essential. The applications of this method are numerous and constantly increasing.

Applications in Microbiology

dPCR was extensively used during the COVID-19 pandemic and continues to be applied for detection of SARS-CoV-2, quantification of viral load, mutation detection, and monitoring of disease progression [9]. Besides COVID-19, assays have been used to quantify many viruses, including HIV DNA and HIV two-long terminal repeat (2-LTR) circles [10,11], CMV [12,13], hepatitis B virus [14], JC polyomavirus [15], human papillomavirus [16], HIV RNA [17- 19], human T-lymphotropic virus [17,18], human rhinoviruses [20], hepatitis C virus [19], hepatitis E virus [21], human parechovirus type 3 [22]. These assays have also been applied for the quantification of Mycobacterium tuberculosis [23], Helicobacter pylori [24] bacterial targets, and the malaria parasite [25]. The utility of dPCR is so substantial that you can use this technique not only for diagnosis, but also for the monitoring of these microbes in the community, by allowing the massive sample processing [26].

Applications in Oncology

Its application in liquid biopsy may represent the most promising clinical use of using dPCR for diagnosis and prognosis and this is evidenced by many different research efforts for a variety of malignancies. Research showed that levels of MACC1 and S100A4 gene derivatives were found to be elevated in ovarian cancer patients compared to healthy subjects (318 serum samples from 79 patients quantified by RT-qPCR and ddPCR) [27]. The increased levels of MACC1 and S100A4 were associated with an increased level of FIGO (advanced gynaecological cancer) and therefore the serological levels of these derivatives can be associated with early diagnosis, overall survival, but also evaluation of the effectiveness of CMT-type methods, surgical cytoreduction with quantitative measurement in serum of patients before and after the use of such methods [28].

Even in types of malignancy with great genetic heterogeneity, such as triple negative, or HER-2 NEG breast cancer, liquid biopsy with dPCR showed tremendous diagnostic benefit, as shown in a study by the Curie Institute of Dr. Carausu’s team in patients with metastatic breast cancer of type HER-2 NEG that began a few years ago-2019. Specifically, in phase III, which includes 1000 patients from over 80 diagnostic centers with HER-2 negative metastatic breast cancer who have undergone endocrine therapy, mutations in genes associated with resistance to each treatment are detected using liquid biopsy and sample processing with dPCR [29]. A basic example is the detection with the help of dPCR real-time mutations in the ESR1 gene in thousands of ctDNA samples (circulating in the blood cancer DNA) before and after endocrine therapy or even after treatment change, an indicator that can be used as a prognostic or diagnostic biomarker [30].

Similar results have shown its use regarding the direct amount of EGFR variants from ctDNA in blood samples from non-small cell lung cancer in a plethora of studies, for example, Wang et al, in which dPCR was used for EGFR T790 gene detection. Clinically, it was shown that T790M‐positive patients have better clinical outcomes to EGFR‐TKIs than T790M‐negative patients, using it as a possible biomarker [31]. Even in cases where tumour cell purity and cellularity are significantly low, dPCR is sensitive enough to detect variants with low allele frequencies that are critical for determining malignancy (such as KRAS), identifying microRNAs (miRNAs), somatic variants, copy number variants, and methylation [32]. Renal cell carcinoma also falls into this category where Sequiera et al. managed to create a ddPCR-based panel to detect 4 circulating mi-RNAs (free RNAs with small amount nucleotides responsible for gene regulation and function). Renal carcinoma patients disclosed significantly higher circulating levels of hsa-miR-155-5p compared to healthy donors, whereas the opposite was observed for hsa-miR-21-5p levels. Furthermore, hsa-miR-21-5p and hsa-miR-155-5p panels detected RCC with high sensitivity (82.66%) and accuracy (71.89%). The hsa-miR-126-3p/hsa-miR-200b-3p panel identified the most common RCC subtype (clear cell, ccRCC) with 74.78% sensitivity [33].

Applications in Genetics and Prenatal Testing

This technique has already been used in prenatal testing as a non-invasive test for genetic diseases, utilizing cffDNA (cell-free fetal DNA), easily obtained from a maternal blood sample. Specific autosomal recessive diseases and predominant monogenic disorders in high-risk pregnancies, based on family history, parental carrier status, or fetal ultrasound findings can be diagnosed or monitored via dPCR, which can be used to detect abnormalities even when there is a small amount of DNA with low variation frequencies.

Firstly, fetal chromosomal aneuploidies can be detected with DPCR; Fan et al. estimated that the sensitivity of cdPCR is much higher than RT-PCR and fluorogenic quantitative PCR in the detection of fetuses with trisomy syndrome 21. In another study that looked at 283 clinical samples, non-invasive prenatal dPCR testing for trisomy 13, 18, and 21 in a single-tube assay showed 100% detection sensitivity and 95.12% specificity [34 – 36].

Second, in autosomal recessive disorders, dPCR has shown its strength early on since 2008, when dPCR was used to detect beta-thalassemia. Quantifying the mutated DNA of the mother and fetus could help diagnose pathogenic fetal genes [37,38]. Many years later, in 2016, Lee et al. detected both common and rare deletions in thalassemia using dPCR [39]. Even sickle cell disease (82% of male fetuses and 75% of female fetuses were diagnosed with SCD using DYS14, the specific marker of the Y chromosome marker) (HBB p.Glu6Val) [40] or spinal muscular atrophy (SMN1 and SMN2 with CVs of 1.7-3.7% and 2.1-2.7%, respectively) have been applied to prenatal screening using dPCR [41].

Comparative Performance of dPCR and Advanced Molecular Diagnostic Technologies

In the context of clinical diagnostics, dPCR has emerged as a valuable alternative and complement to traditional methods such as qPCR and next-generation sequencing (NGS). Compared to qPCR, dPCR offers superior sensitivity and precision, particularly in detecting low-abundance targets. This is critical in infectious disease diagnostics, where dPCR has demonstrated improved performance over qPCR in identifying low viral loads of pathogens such as SARS-CoV-2, often detecting cases missed by qPCR due to its lower limit of detection and greater tolerance to inhibitors [42, 43].

In oncology, dPCR outperforms qPCR in quantifying circulating tumor DNA (ctDNA) mutations with greater accuracy and reproducibility, making it especially suited for monitoring minimal residual disease and treatment response [44]. While NGS offers unparalleled breadth for mutation discovery and comprehensive profiling, dPCR excels in the targeted quantification of known variants. For instance, studies comparing ddPCR and NGS in detecting EGFR mutations in non-small cell lung cancer (NSCLC) report concordance rates above 90%, highlighting dPCR’s utility as a reliable and cost-effective follow-up tool for high-throughput genomic analysis [45, 46]. Moreover, dPCR’s faster turnaround time and simpler workflow compared to NGS make it an attractive choice for clinical settings requiring rapid, actionable results.

Emerging innovations are rapidly extending the clinical potential of dPCR. The advent of CRISPR-dPCR combines CRISPR-based sequence recognition with digital partitioning, enabling ultra-high specificity and sensitivity. For example, RADICA (Rapid DIgital Crispr Approach) has achieved absolute quantification of SARS-CoV-2 RNA in under an hour, four times faster than traditional dPCR, with equivalent accuracy, paving the way for rapid, precise viral diagnostics in clinical settings [47].

Parallel strides in point-of-care (POC) integration are transforming dPCR into portable, user-friendly formats. A smartphone-operated handheld dPCR device (“SPEED”) demonstrates that thermal cycling, fluorescence detection, and data analysis can be miniaturized into a low-cost unit, with performance comparable to benchtop systems [48]. Such platforms are well-positioned for decentralized testing, a crucial requirement during infectious outbreaks or in resource-limited clinics.

In single-cell applications, microfluidic-based dPCR offers transformative insights into cellular heterogeneity. Reviews highlight its utility in quantifying gene expression and rare mutations from individual cells with high precision and throughput. In viral diagnostics, single-cell ddPCR has been used to identify HIV- or HBV-infected cells without DNA extraction, techniques that hold significant promise for early infection detection and therapy monitoring [49, 50].

These developments position dPCR as an adaptable technique that complements existing tools. CRISPR-dPCR excels in ultra-sensitive, rapid detection of known targets; POC systems bring this capability to the bedside or field; and single-cell dPCR addresses cellular-level diagnostics in oncology, immunology, and prenatal care. As these formats mature and accrue rigorous clinical validation, dPCR stands to play a pivotal role across precision medicine, infectious disease management, and point-of-care diagnostics.

Future Perspectives and Challenges

Despite its advantages, dPCR faces several challenges that hinder its widespread clinical adoption. One of the primary concerns is the high cost associated with dPCR instruments and reagents, which limits accessibility in resource-constrained settings. Additionally, there is a need for standardized protocols and regulatory guidelines to ensure consistency and reliability across different laboratories and clinical applications. The complexity of data analysis and interpretation also presents a barrier, necessitating the development of user-friendly software and bioinformatics tools to streamline workflows. Ongoing advancements in automation aim to address these limitations by enhancing the efficiency and scalability of dPCR platforms. The integration of microfluidics and droplet-based technologies has led to the development of high-throughput dPCR systems capable of processing large sample volumes with reduced reagent consumption. Furthermore, efforts to optimize assay designs and improve multiplexing capabilities are expected to expand the utility of dPCR in clinical diagnostics. The incorporation of dPCR into routine clinical practice also requires robust validation studies to demonstrate its clinical utility and cost-effectiveness compared to existing molecular techniques. As emerging research continues to refine dPCR methodologies, its potential to become a mainstream diagnostic tool will largely depend on collaborations between researchers, healthcare providers, and industry stakeholders to address these challenges and drive widespread implementation.

CONCLUSION

dPCR represents a transformative advancement in molecular diagnostics, offering exceptional precision, sensitivity, and the ability to provide absolute quantification of nucleic acids. Its proven utility in infectious disease diagnostics, oncology, and prenatal testing underscores its potential to reshape clinical decision-making. Compared to conventional PCR methods, dPCR demonstrates superior performance in detecting low-abundance targets, overcoming inhibitors, and enabling multiplex analysis in complex samples. However, despite its considerable promise, dPCR is not without limitations. High costs, limited scalability for high-throughput applications, and current constraints in multiplexing capacity pose significant barriers to widespread clinical adoption [51]. Moreover, the lack of standardized protocols and regulatory guidelines hinders integration into routine diagnostics and limits inter-laboratory comparability. Reducing operational costs and developing robust clinical validation studies will be essential for mainstream implementation. Interdisciplinary collaboration among clinicians, researchers, and regulatory bodies is also critical to establish evidence-based guidelines and ensure that dPCR transitions effectively from a specialized research tool into a routine component of precision medicine. With continued innovation and systemic support, dPCR holds strong potential to become a cornerstone in next-generation diagnostics, ultimately improving patient care and clinical outcomes.

Conflict of interest

None to declare

Declaration of funding sources

None to declare

Author contributions statement

AP and ZA were responsible for the narrative review analysis; AP and ZA were responsible for drafting the manuscript; AV was responsible for the revision of the manuscript; all authors provided final approval for the version to be submitted.

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