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.

数字聚合酶链反应 (dPCR) 技术可以使用基于液滴或基于芯片的系统量化脱氧核糖核酸的特定序列。单个荧光通道中的 dPCR 双工方法通常基于两个目标之间的荧光振幅 ( F ) 的差异。使用不同的探针浓度或不同的目标长度，通过F值变化来区分不同的目标。在本研究中，我们提出了一种基于单一荧光通道的 dPCR 双工方法，该方法结合了特定的探针和嵌入染料以增加F的差异两个目标之间的值。我们选择了两个序列，一个来自 18 号染色体 (Chr18)，仅由嵌入染料 EvaGreen 检测，另一个来自 21 号染色体 (Chr21)，由 6-羧基荧光素 (FAM) 探针和 EvaGreen 联合检测。我们执行了 dPCR 协​​议并在室温下对 dPCR 芯片进行了成像，以验证所提出的双工方法。结果表明，与仅使用 EvaGreen 检测两种扩增子相比，使用 FAM 探针检测 Chr21 时，Chr18 和 Chr21 之间的F值差异从≈5% 增加到 20%。添加的 FAM 探针使用单色荧光通道启用双目标识别。我们进一步确定了不同温度下F值的差异使用人工 dPCR 图像。这种提议的方法代表了单荧光通道 dPCR 双工的简单选择，使其适用于用于即时应用的简化 dPCR 系统。