Multiplex Detection of Nucleic Acids Using Recombinase Polymerase Amplification and a Molecular Colorimetric...

Nucleic acid analysis has become highly relevant for point-of-care (POC) diagnostics since the advent of isothermal amplification methods that do not require thermal cycling. In particular, recombinase polymerase amplification (RPA) combined with lateral flow detection offers a rapid and simple solution for field-amenable low-resource nucleic acid testing. Expanding POC nucleic acid tests for the detection of multiple analytes is vital to improve diagnostic efficiency because increased multiplexing capacity enables higher information density combined with reduced assay time and costs. Here, we investigate expanding RPA POC detection by identifying a generic multiplex RPA format that can be combined with a generic multiplex lateral flow device (LFD) to enable binary and molecular encoding for the compaction of diagnostic data. This new technology relies on the incorporation of molecular labels to differentiate nucleic acid species spatially on a lateral flow membrane. In particular, we identified additional five molecular labels that can be incorporated during the RPA reaction for subsequent coupling with LFD detection. Combined with two previously demonstrated successful labels, we demonstrate potential to enable hepta-plex detection of RPA reactions coupled to multiplex LFD detection. When this hepta-plex detection is combined with binary and molecular encoding, an intuitive 7-segment output display can be produced. We note that in all experiments, we used an identical DNA template, except for the 5' label on the forward primer, to eliminate any effects of nucleic acid sequence amplification bias. Our proof-of-concept technology demonstration is highly relevant for developing information-compact POC diagnostics where space and time are premium commodities.