Digital polymerase chain reaction duplexing method in a single fluorescence channel

Melt‐Encoded‐Tags for Expanded Optical Readout in Digital PCR (METEOR‐dPCR) Enables Highly Multiplexed Quantitative Gene Panel Profiling

“…[ 9 ] Secondly, multiplexing in dPCR is limited by the number of fluorescence channels in the instrument. Although to some extent the multiplexing capacity can be extended by using melt characteristics, [ 10 ] kinetic (amplification curve) analysis, [ 11 ] incorporating probes with different fluorescence intensities, [ 12 ] or even applying deep learning to distinguish probes with overlapping fluorescence spectrum, [ 13 ] the need for large number of fluorescence probes increase the cost, complexity and potential for nonspecific signals. Due to these challenges, a scalable and practical multitarget assay based on dPCR has not been realized.…”

Similar color analysis based on deep learning (SCAD) for multiplex digital PCR via a single fluorescent channel

“…, applying probes with photobleaching dyes 18 or using an intercalating dye for two targets and adding another specific fluorescence probe to one of the targets. 19 However, there are still several challenges associated with these methods to achieve multiplex detection within a single channel. First, the multiplex reaction system needs careful optimization to avoid interference from the ‘rain’ phenomenon.…”

“…To address this issue, some teams encode the final fluorescence intensities for different targets, such as encoding different concentration probes for different targets 17 or building a dPCR system with special probes and fluorescent dyes to increase the signal difference between multiple targets, e.g., applying probes with photobleaching dyes 18 or using an intercalating dye for two targets and adding another specific fluorescence probe to one of the targets. 19 However, there are still several challenges associated with these methods to achieve multiplex detection within a single channel. First, the multiplex reaction system needs careful optimization to avoid interference from the 'rain' phenomenon.…”