The digital polymerase chain reaction (dPCR) technique can quantify specific sequences of deoxyribonucleic acid using either a droplet-based or chip-based system. dPCR duplexing methods in a single fluorescence channel are typically based on the difference in fluorescence amplitude (F) between two targets. The different targets are distinguished from each other by the F-value variation using non-equal probe concentrations or different target lengths. In the present study, we propose a single fluorescence channel-based dPCR duplexing method that combines a specific probe and intercalating dye to increase the difference in F values between the two targets. We selected two sequences, one from chromosome 18 (Chr18) detected only by the intercalating dye EvaGreen and the other from chromosome 21 (Chr21) detected by a combination of a 6-carboxyfluorescein (FAM) probe and EvaGreen. We performed the dPCR protocol and imaged the dPCR chip at room temperature to verify the proposed duplexing method. The result revealed that the difference in F values between Chr18 and Chr21 increased from ≈5% to 20% when using the FAM probe for Chr21 compared with the detection of both amplicons using EvaGreen only. The added FAM probe enabled two-target discrimination using a single-color fluorescent channel. We further determined the difference in F values at different temperatures using artificial dPCR images. This proposed method represents a simple option for single fluorescence channel dPCR duplexing, making it suitable for simplified dPCR systems used for point-of-care applications.

Central European Institute of Technology Brno University of Technology Purkyňova 123 612 00 Brno Czech Republic

Department of Microsystem Engineering School of Mechanical Engineering Northwestern Polytechnical University 127 West Youyi Road Xi'an Shaanxi 710072 PR China

Department of Microsystem Engineering School of Mechanical Engineering Northwestern Polytechnical University 127 West Youyi Road Xi'an Shaanxi 710072 PR China; The Key Laboratory of Biomedical Information Engineering of Ministry of Education School of Life Science and Technology Xi'an Jiaotong University Xi'an 710049 PR China

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Albertov 4 128 00 Prague Czech Republic

Institute of Biology and Medical Genetics 1st Faculty of Medicine Charles University and General University Hospital Prague Albertov 4 128 00 Prague Czech Republic; Department of Laboratory Medicine Faculty of Health Care and Social Work University of Trnava in Trnava Universitne namestie 1 918 43 Trnava Slovak Republic

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