Digital PCR versus Quantitative PCR in Diagnostic Pathology: Principles, Clinical Applications, Validation, and Practical Workflow Recommendations — A Comparative Review
DOI:
https://doi.org/10.63501/hrm56e02Keywords:
digital PCR; quantitative PCR; diagnostic pathology; molecular diagnostics; liquid biopsy; minimal residual disease; assay validation; copy number variation.Abstract
Polymerase chain reaction (PCR) technologies are foundational to modern diagnostic pathology. Quantitative PCR (qPCR) has long served as the clinical standard for nucleic acid detection and relative quantification, while digital PCR (dPCR) introduces enhanced analytical precision and sensitivity, particularly for low-abundance targets. This comparative review evaluates mechanistic differences, analytical performance, and clinical applications of qPCR and dPCR, with recommendations for validation and workflow optimization in diagnostic laboratories. Structured analysis demonstrates that both technologies occupy complementary roles in precision molecular pathology. Expanded discussion of validation metrics, clinical workflow, and practical recommendations provides a framework for integration of these technologies into modern diagnostic practice.
References
1) Mackay IM, Arden KE, Nitsche A. Real-time PCR in virology. Nucleic Acids Res. 2002;30:1292–1305.
2) Hindson BJ, Ness KD, Masquelier DA, Belgrader P, Heredia NJ, Makarewicz AJ, Bright IJ, Lucero MY, Hiddessen AL, Legler TC, Kitano TK. High-throughput droplet digital PCR system for absolute quantitation of DNA copy number. Analytical chemistry. 2011 Nov 15;83(22):8604-10.
3) Whale, A. S., Huggett, J. F., Cowen, S., Speirs, V., Shaw, J., Ellison, S., Foy, C. A., & Scott, D. J. (2012). Comparison of microfluidic digital PCR and conventional quantitative PCR for measuring copy number variation. Nucleic acids research, 40(11), e82. https://doi.org/10.1093/nar/gks203.
4) Huggett JF, Foy CA, Benes V, Emslie K, Garson JA, Haynes R, Hellemans J, Kubista M, Mueller RD, Nolan T, Pfaffl MW. The Digital MIQE Guidelines: M inimum I nformation for Publication of Q uantitative Digital PCR E xperiments. Clinical chemistry. 2013 Jun 1;59(6):892-902.
5) Beaver JA, Jelovac D, Balukrishna S, Cochran RL, Croessmann S, Zabransky DJ, Wong HY, Valda Toro P, Cidado J, Blair BG, Chu D. Detection of cancer DNA in plasma of patients with early-stage breast cancer. Clinical cancer research. 2014 May 15;20(10):2643-50.
6) Heredia, N. J., Belgrader, P., Wang, S., Koehler, R., Regan, J., Cosman, A. M., Saxonov, S., Hindson, B., Tanner, S. C., Brown, A. S., & Karlin-Neumann, G. (2013). Droplet Digital™ PCR quantitation of HER2 expression in FFPE breast cancer samples. Methods (San Diego, Calif.), 59(1), S20–S23. https://doi.org/10.1016/j.ymeth.2012.09.012.
7) Strain MC, Lada SM, Luong T, Rought SE, Gianella S, Terry VH, Spina CA, Woelk CH, Richman DD. Highly precise measurement of HIV DNA by droplet digital PCR. PloS one. 2013 Apr 3;8(4):e55943..
8) Oxnard GR, Paweletz CP, Kuang Y, Mach SL, O'Connell A, Messineo MM, Luke JJ, Butaney M, Kirschmeier P, Jackman DM, Jänne PA. Noninvasive detection of response and resistance in EGFR-mutant lung cancer using quantitative next-generation genotyping of cell-free plasma DNA. Clinical cancer research. 2014 Mar 15;20(6):1698-705.
9) Drandi, D., Kubiczkova-Besse, L., Ferrero, S., Dani, N., Passera, R., Mantoan, B., Gambella, M., Monitillo, L., Saraci, E., Ghione, P., Genuardi, E., Barbero, D., Omedè, P., Barberio, D., Hajek, R., Vitolo, U., Palumbo, A., Cortelazzo, S., Boccadoro, M., Inghirami, G., … Ladetto, M. (2015). Minimal Residual Disease Detection by Droplet Digital PCR in Multiple Myeloma, Mantle Cell Lymphoma, and Follicular Lymphoma: A Comparison with Real-Time PCR. The Journal of molecular diagnostics : JMD, 17(6), 652–660. https://doi.org/10.1016/j.jmoldx.2015.05.007.
10) Dong L, Meng Y, Sui Z, Wang J, Wu L, Fu B. Comparison of four digital PCR platforms for accurate quantification of DNA copy number of a certified plasmid DNA reference material. Scientific reports. 2015 Aug 25;5(1):13174..
Downloads
Published
Issue
Section
License
This article is licensed under a Creative Commons Attribution 4.0 International License (CC BY 4.0), which permits use, sharing, adaptation, distribution, and reproduction in any medium or format, provided appropriate credit is given to the author(s) and the source. To view a copy of this license, visit: https://creativecommons.org/licenses/by/4.0