Multicolor Bioluminescent Toolbox for Multiplex Glow-in-the-Dark Nucleic Acid Diagnostics

Combining bioluminescent sensors and isothermal amplification enables fast and simple nucleic acid diagnostics, holding great potential for point-of-care applications. We previously developed the LUNAS (luminescent nucleic acid sensor) platform, which integrates recombinase polymerase amplification (RPA) with a clustered regularly interspaced short palindromic repeats (CRISPR) dCas9-mediated bioluminescent readout. Here, we extend LUNAS into a multiplex platform through the development of green- and red-light emitting LUNAS variants. These color variants can be integrated with duplex RPA for amplification and orthogonal detection of two DNA targets in a single reaction, without compromising sensitivity or reaction speed. Using this approach, we developed a duplex assay for the sexually transmitted infections gonorrhea and chlamydia, with attomolar sensitivity and a robust ratiometric readout. Using a simple digital camera, multicolor LUNAS enabled detection of bacterial DNA from clinical samples with loads down to ∼100 copies/μL and within ∼30 min, demonstrating its diagnostic performance for low-resource applications.