RUNCOV : a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2 Robène, I. Jouen, E. Maillot-Lebon, V. Fenelon, B. T. Hascoat, J. Pecrix, Y. /Richard, Damien Jaffar-Bandjee, M. C. Mavingui, P. Chiroleu, F. Becker, N. Poubeau, P. Ramiandrisoa, M. Sin, M. Costet, L. Laurent, A. Laurent, P. Chabirand, A. Moreau, A. Reynaud, B. Jeuffrault, E. Cetre-Sossah, C. SARS-CoV-2 triplex-RT-LAMP detection point-of-care testing 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. 2025 text https://www.documentation.ird.fr/hor/fdi:010092872 fdi:010092872 Robène I., Jouen E., Maillot-Lebon V., Fenelon B. T., Hascoat J., Pecrix Y., Richard Damien, Jaffar-Bandjee M. C., Mavingui P., Chiroleu F., Becker N., Poubeau P., Ramiandrisoa M., Sin M., Costet L., Laurent A., Laurent P., Chabirand A., Moreau A., Reynaud B., Jeuffrault E., Cetre-Sossah C.. RUNCOV : a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2. 2025, 10 (1), bpaf010 [16 p.] EN