Luminescent proteins to diagnose viral diseases

Despite recent advances, many of the highly sensitive diagnostic tests that can detect viral diseases still require complicated procedures to prepare a sample or interpret a result and are therefore impractical to perform. carry them out in direct medical care centers or in low-income areas. A recent advance may substantially change this situation.

Scientists have developed a sensitive method that analyzes viral nucleic acids in just 20 minutes and can be completed in a single step using ‘glow-in-the-dark’ proteins.

The flash of the firefly, the glow of the Lophiiformes lure, and the ghostly blue of phytoplankton-covered beaches all feed on the same scientific phenomenon known as bioluminescence. The luminescent effect, which causes them to glow in the dark, is caused by a chemical reaction involving the luciferase protein. This protein has been incorporated into sensors that, when they detect their target, emit an easily observable light. Because of their simplicity, these types of sensors are ideal for point-of-care testing, but for clinical diagnostic testing, they need incredibly high sensitivity that has previously not been achievable. That capability could be achieved by the gene editing technique known as Clustered Regularly Spaced Short Palindromic Repeats (CRISPR), except that it requires many additional steps and specialized equipment to be able to detect a possible signal. low in a complex and chaotic sample. So Maarten Merkx’s team at Eindhoven University of Technology in the Netherlands wanted to use CRISPR-associated proteins, but combine them with a technique using bioluminescence whose signal could be detected with a simple digital camera.

To ensure they had enough sample RNA or DNA to test, the researchers used the recombinase polymerase amplification (RPA) technique, a simple method that operates at a constant temperature of approximately 37 degrees. centigrade Using the new system, called luminescent nucleic acid sensor (LUNAS), it is achieved that each of the two specific CRISPR/Cas9 proteins from the different neighboring parts of a viral genome binds to a different fragment. of luciferase. In the event that the specific viral genome researchers are looking for is present, the two CRISPR/Cas9 proteins bind to the target nucleic acid sequences and move closer to each other, allowing a complete protein to be formed. luciferase that, in the presence of a chemical substrate, emits a blue light. To confirm that such a substrate is being used, a control reaction that glows green is used. If the green tube turns blue, the result is positive.

Thanks to proteins that have a blue or green glow, like the ones described here, the diagnosis of a disease could be easier and faster. (Image: Maarten Merkx)

When the study authors tested clinical samples collected using nasal swabs, the RPA-LUNAS successfully detected SARS-CoV-2 RNA within 20 minutes, even at concentrations as low as 200 copies per minute. microliter. The researchers say the LUNAS assay has great potential to detect many other viruses efficiently and easily.

The study is titled “Glow-in-the-Dark Infectious Disease Diagnostics Using CRISPR-Cas9-Based Split Luciferase Complementation”. And it has been published in the academic journal ACS Central Science. (Source: ACS)