Bioluminescence is the emission of light by a living organism. A chemical reaction involving the luciferase protein causes this luminescent, “glow-in-the-dark” effect. The luciferase protein can also be incorporated into sensors that emit light in the presence of their target. Such sensors are simple, which makes them ideal for point-of-care testing. However, so far, they often have shown insufficient sensitivity for a clinical diagnostic test.
Maarten Merkx, Eindhoven University of Technology, The Netherlands, and colleagues have developed a “glow-in-the-dark” assay that enables the sensitive detection of pathogen DNA/RNA within 20 min. The approach combines a (bio)luminescent nucleic acid sensor (LUNAS), which is based on CRISPR-mediated luciferase activation, with recombinase polymerase amplification (RPA), which improves the sensitivity.
To make sure there is enough sample RNA or DNA to analyze, the researchers first performed RPA, which operates at a constant temperature of about 40 °C. The amplified samples are then analyzed using LUNAS. For this, two CRISPR/Cas9 proteins specific for different neighboring parts of a viral genome each have a distinct fragment of luciferase attached to them. If the targeted viral genome is present, the two CRISPR/Cas9 proteins bind to the targeted nucleic acid sequences and come close to each other, allowing the complete luciferase protein to form and emit light.
When tested on clinical samples collected from nasal swabs, the RPA-LUNAS method successfully detected SARS-CoV-2 RNA within 20 minutes, even at low concentrations. According to the researchers, RPA-LUNAS is attractive for point-of-care infectious disease testing.
- Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation,
Harmen J. van der Veer, Eva A. van Aalen, Claire M. S. Michielsen, Eva T. L. Hanckmann, Jeroen Deckers, Marcel M. G. J. van Borren, Jacky Flipse, Anne J. M. Loonen, Joost P. H. Schoeber, Maarten Merkx,
ACS Cent. Sci. 2023.