Virtual Fluorescence Color Channels by Selective Photobleaching in Digital PCR Applied to the Quantification of KRASPoint Mutations

Multiplexing of analyses is essential to reduce sample and reagent consumption in applications with large target panels. In applications such as cancer diagnostics, the required degree of multiplexing often exceeds the number of available fluorescence channels in polymerase chain reaction (PCR) devices. The combination of photobleaching-sensitive and photobleaching-resistant fluorophores of the same color can boost the degree of multiplexing by a factor of 2 per channel. The only additional hardware required to create virtual fluorescence color channels is a low-cost light-emitting diode (LED) setup for selective photobleaching. Here, we present an assay concept for fluorescence color multiplexing in up to 10 channels (five standard channels plus five virtual channels) using the mediator probe PCR with universal reporter (UR) fluorogenic oligonucleotides. We evaluate the photobleaching characteristic of 21 URs, which cover the whole spectral range from blue to crimson. This comprehensive UR data set is employed to demonstrate the use of three virtual channels in addition to the three standard channels of a commercial dPCR device (blue, green, and red) targeting cancer-associated point mutations (KRASG12D and G12V). Moreover, a LOD (limit of detection) analysis of this assay confirms the high sensitivity of the multiplexing method (KRASG12D: 16 DNA copies/reaction in the standard red channel and KRASG12V: nine DNA copies/reaction in the virtual red channel). Based on the presented data set, optimal fluorogenic reporter combinations can be easily selected for the application-specific creation of virtual channels, enabling a high degree of multiplexing at low optical and technical effort.