Historically, disease detection and treatment in humans relied on serological and culture-based methods. While these techniques were inexpensive and easy to perform, they came with limitations, especially in terms of sensitivity and early detection.
Related Products
Evolution of Diagnostic Methods
Serological methods, utilizing blood, saliva, and tissue tests, have been traditional approaches for assessing disease risks. However, these methods have limitations, prompting the need for more precise and early detection techniques. Molecular-based detection technologies, such as polymerase chain reaction (PCR) and quantitative PCR (qPCR), have become widely employed for detecting viruses and tumors. PCR, in particular, rapidly amplifies DNA segments for in-depth analysis.
Generations of PCR
PCR has undergone evolution, progressing through three generations. The first generation relied on gel electrophoresis and had limitations in terms of time consumption and sensitivity. The second generation, real-time quantitative PCR (RT-qPCR), improved quantification but faced challenges with interference substances. The third generation, Digital PCR (dPCR), introduced absolute quantification through reaction partitioning, providing high sensitivity and accuracy.
Introduction of Digital PCR
Digital PCR (dPCR) stands as a paradigm-shifting achievement in the realm of molecular biology, presenting an exceptionally sensitive and precise methodology for quantifying nucleic acids. The genesis of dPCR traces back to the early 1990s, when Skyes et al. pioneered the concept of absolute measurement, employing limiting dilution and end-point signal quantification. Their focus on identifying genes with mutated heavy chains set the stage for a transformative approach. Nevertheless, the definitive terminology "digital PCR" in its contemporary context emerged in 1999, propelled by the groundbreaking work of Vogelstein and Kinzler. Their method involved intricate processes of sample dilution, partitioning, and subsequent signal amplification and detection facilitated through fluorescence.
Fig. 1 The basic principle of digital polymerase chain reaction (Nazir S., 2023).
As time has progressed, dPCR methodologies have evolved and become commercially accessible, integrating technologies such as microfluidic chips, discs, arrays, and microdroplets based on oil-water emulsions. The core principle of dPCR revolves around dividing the sample into numerous small compartments, each containing zero, one, or more molecules. After amplification, the fluorescence intensity in each compartment is analyzed, allowing precise and absolute quantification. The term "digital" in dPCR accurately reflects the binary nature of the signal-either on or off, fluorescent or non-fluorescent, activated or not. This three-step process of sample dispersion, amplification, and signal monitoring forms the essence of dPCR, enabling the detection of minute quantities of target genes with unparalleled sensitivity.
Advantages and Disadvantages of dPCR
Digital PCR (dPCR) has emerged as a preferred choice in diverse biomedical applications, owing to its distinctive advantages. One key strength lies in its ability to partition the reaction mixture into numerous compartments. This strategic partitioning mitigates the impact of inhibitors and significantly enhances the signal-to-noise ratio. Consequently, this feature contributes to heightened sensitivity and precise quantification, setting dPCR apart from quantitative PCR (qPCR). Unlike qPCR, dPCR eliminates the dependency on reference materials for quantification. This independence imparts greater robustness and reproducibility to the technique. A noteworthy advantage is the multiplexing capability of dPCR, allowing for the simultaneous detection of multiple targets.
However, like any technological innovation, dPCR does have its limitations. Traditional qPCR instruments exhibit a broader capacity for detecting fluorescence signals from various dyes compared to dPCR, thereby limiting the latter's multiplexing potential. Despite its high sensitivity, the application of dPCR may be constrained by the necessity for specialized equipment and reagents. Furthermore, inherent in any molecular detection technique, including dPCR, is the risk of contamination. Addressing these challenges is currently a focal point of ongoing research and development within the field.
Applications of dPCR
Digital PCR (dPCR) stands out for its versatility and efficacy, finding application across diverse fields. In cancer detection, dPCR excels in accurately, sensitively, and reproducibly quantifying circulating tumor DNA (ctDNA). This non-invasive method holds immense potential for early cancer diagnosis, monitoring treatment effectiveness, and assessing recurrence risk. DPCR's capability to identify rare genetic mutations, such as those associated with breast cancer, positions it as a valuable asset in personalized medicine.
Digital PCR (dPCR) makes a significant contribution to non-invasive prenatal diagnosis, showcasing its ability to identify genetic anomalies in cell-free fetal DNA from maternal plasma. This positions dPCR as a promising technology for prenatal screening, particularly for conditions like trisomy 21. The affordability and accessibility of dPCR make it a practical choice for widespread screening programs.
In infectious diseases, dPCR establishes itself as a reliable tool for virus detection. Its noteworthy performance in detecting the SARS-CoV-2 virus during the COVID-19 pandemic emphasizes dPCR's superiority in sensitivity and accuracy, especially in samples with low viral loads. Beyond COVID-19, dPCR effectively detects viruses such as cytomegalovirus (CMV), HIV, and influenza, offering heightened sensitivity and precision compared to conventional methods.
In summary, dPCR's applications are expanding, driven by its unparalleled sensitivity, precision, and capacity for absolute quantification. Whether applied to cancer diagnostics, infectious disease detection, or prenatal screening, dPCR stands at the forefront of molecular detection technologies, providing researchers and clinicians with a potent tool for advancing medical science.
References
- Nazir S. Medical diagnostic value of digital PCR (dPCR): A systematic review. Biomedical Engineering Advances. 2023: 100092.
- Zhang L., et al. Emerging digital PCR technology in precision medicine. Biosensors and Bioelectronics. 2022, 211: 114344.
.
