Digital polymerase chain reaction duplexing method in a single fluorescence channel

• Published in: Analytica chimica acta Vol. 1238; p. 340243
• Main Authors: Zhang, Haoqing, Laššáková, Soňa, Yan, Zhiqiang, Wang, Xinlu, Šenkyřík, Pavel, Gaňová, Martina, Chang, Honglong, Korabečná, Marie, Neuzil, Pavel
• Format: Journal Article
• Language: English
• Published: Elsevier B.V 15.01.2023
• Subjects: Absolute quantification; Digital polymerase chain reaction; Duplexing methods; Single fluorescence channel duplexing; Duplexing methods; Digital polymerase chain reaction; Single fluorescence channel duplexing; Absolute quantification
• ISSN: 0003-2670; 1873-4324; 1873-4324
• DOI: 10.1016/j.aca.2022.340243

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. We proposed a single fluorescence channel-based dPCR duplexing method combining a specific probe and intercalating dye to distinguish two targets based on the difference in fluorescent amplitudes. It simplified dPCR systems used for point-of-care applications. [Display omitted] •A method of single fluorescent channel dPCR multiplexing•Fluorescence value separation of two targets by using intercalating dye for both and adding specific FAM probe to one.•Suitable for simplified dPCR systems for point of care applications.•The image capture of the dPCR chip is conducted at ambient temperature.