The digital polymerase chain reaction (dPCR) multiplexing method can simultaneously detect and quantify closely related deoxyribonucleic acid sequences in complex mixtures. The dPCR concept is continuously improved by the development of microfluidics and micro- and nanofabrication, and different complex techniques are introduced. In this review, we introduce dPCR techniques based on sample compartmentalization, droplet- and chip-based systems, and their combinations. We then discuss dPCR multiplexing methods in both laboratory research settings and advanced or routine clinical applications. We focus on their strengths and weaknesses with regard to the character of biological samples and to the required precision of such analysis, as well as showing recently published work based on those methods. Finally, we envisage possible future achievements in this field.

1st Faculty of Medicine Institute of Biology and Medical Genetics Charles University and General University Hospital 12800 Prague Czech Republic

Central European Institute of Technology Brno University of Technology 612 00 Brno Czech Republic

Central European Institute of Technology Brno University of Technology 612 00 Brno Czech Republic; School of Mechanical Engineering Northwestern Polytechnical University Xi'an 710072 PR China; The Faculty of Electrical Engineering and Communication Brno University of Technology 616 00 Brno Czech Republic

School of Mechanical Engineering Northwestern Polytechnical University Xi'an 710072 PR China

References provided by Crossref.org

SPEED: an integrated, smartphone-operated, handheld digital PCR Device for point-of-care testing

Temperature non-uniformity detection on dPCR chips and temperature sensor calibration

Design considerations for point-of-need devices based on nucleic acid amplification for COVID-19 diagnostics and beyond

Determination of Advantages and Limitations of qPCR Duplexing in a Single Fluorescent Channel