Although there have been significant advancements in diagnostic tests for viral diseases, many highly sensitive tests still rely on complex sample preparation and result interpretation methods, rendering them unsuitable for point-of-care settings or resource-limited areas. However, in the journal ACS Central Science, researchers have now revealed a novel, sensitive technique that can analyze viral nucleic acids in just 20 minutes using a one-step process with “glow-in-the-dark” proteins.
Bioluminescence, the scientific phenomenon behind the firefly’s glow, the anglerfish’s radiant lure, and the ghostly blue of phytoplankton-laden shores, is powered by a chemical reaction involving the luciferase protein. This luminescent protein has been integrated into sensors that emit visible light when detecting their target, making them ideal for straightforward point-of-care testing. However, until now, these sensors have not achieved the exceptional sensitivity necessary for clinical diagnostic tests.
The gene-editing technique known as CRISPR could provide this ability, but it requires many steps and additional specialized equipment to detect what can be a low signal in a complex, noisy sample. So, Maarten Merkx and colleagues wanted to use CRISPR-related proteins, but combine them with a bioluminescence technique whose signal could be detected with just a digital camera.
To make sure there was enough sample RNA or DNA to analyze, the researchers performed recombinase polymerase amplification (RPA), a simple method that works at a constant temperature of about 100 F. With the new technique, called LUNAS (luminescent nucleic acid sensor), two CRISPR/Cas9 proteins specific for different neighboring parts of a viral genome each have a distinct fragment of luciferase attached to them.
If a specific viral genome that the researchers were testing for was present, the two CRISPR/Cas9 proteins would bind to the targeted nucleic acid sequences and come close to each other, allowing the complete luciferase protein to form and shine blue light in the presence of a chemical substrate. To account for this substrate being used up, the researchers used a control reaction that shined green. A tube that changed from green to blue indicated a positive result.
When tested on clinical samples collected from nasal swabs, RPA-LUNAS successfully detected SARS-CoV-2 RNA within 20 minutes, even at concentrations as low as 200 copies per microliter. The researchers say that the LUNAS assay has great potential for detecting many other viruses effectively and easily.
Reference: “Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation” by 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 and Maarten Merkx, 15 March 2023, ACS Central Science.
The study was funded by the Dutch Research Council | Nationaal Regieorgaan Praktijkgericht Onderzoek SIA (NRPO-SIA) and the Eindhoven University Fund.