RUNCOV: a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2

• Published in: Biology methods and protocols 2025-02
• Main Authors: Robène, Isabelle, Jouen, Emmanuel, Maillot-Lebon, Véronique, Fenelon, Babbitha T, 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
• Format: Article
• Language: English
• ISSN: 2396-8923; 2396-8923
• DOI: 10.1093/biomethods/bpaf010

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 5-20 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.