RUNCOV: A one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2
2025
Robène, Isabelle | Jouen, Emmanuel | Maillot-Lebon, Marie-Véronique | Fenelon, Babbitha | Hascoat, Jérémy | Pecrix, Yann | Richard, Damien | Jaffar-Bandjee, Marie-Christine | Mavingui, Patrick | Chiroleu, Frédéric | Becker, Nathalie | Poubeau, Patrice | Ramiandrisoa, Mahery | Sin, Michel | Costet, Laurent | Laurent, Annie | Laurent, Philippe | Chabirand, Aude | Moreau, Aurélie | Reynaud, Bernard | Jeuffrault, Eric | Cetre-Sossah, Catherine
Given the risk of zoonotic disease emergence, including new SARS-CoV-2 variants of COVID-19, rapid diagnostic tools are urgently needed to improve the control of the spread of infectious diseases. A one-pot triplex real-time RT-LAMP (reverse-transcription-loop-mediated isothermal amplification) assay, based on two regions of the genome SARS-CoV-2—specifically the Orf1ab and N genes—along with one internal control, the human RNase P gene, was developed. The multiplexing relies on the distinct melting peaks produced during an annealing step. This tool, named RUNCOV, was compared to the gold-standard reverse-transcription real-time quantitative PCR (RT-qPCR) assay. A simple sample preparation step was designed alongside the assay, making it ready for use on site, as a point-of-care diagnostic tool. RUNCOV is rapid (typically less than 40 minutes), highly sensitive and specific. When tested on clinical samples with known SARS-CoV-2 status, its limit of detection (LOD) ranges between 5 and 20 copies per reaction and its diagnostic sensitivity (97.44%) and specificity (100%) values are high compared to the RT-qPCR gold standard. These results were supported with an extensive in silico analysis of over 14 million genomes, demonstrating this tool was capable of detecting all known SARS-CoV-2 variants, including the most recent ones KP.3.1.1 and BA2.86.1. This molecular assay is portable, as demonstrated when it was used successfully in La Réunion in different contexts outside the laboratory.
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