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

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. 2025. RUNCOV: A one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2. Biology Methods and Protocols, 10 (1):bpaf010, 16 p.

Résumé : 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.

Mots-clés Agrovoc : coronavirus 2 du syndrome respiratoire aigu sévère, zoonose, covid-19, PCR, contrôle de maladies, génomique, maladie infectieuse, génome

Mots-clés géographiques Agrovoc : France, La Réunion

Mots-clés libres : SARS-COV-2, Triplex-RT-LAMP, Detection, Point-of-care testing

Agences de financement européennes : European Agricultural Fund for Rural Development, European Regional Development Fund

Agences de financement hors UE : French Government, Conseil Régional de La Réunion, Centre de Coopération Internationale en Recherche Agronomique pour le Développement

Auteurs et affiliations

• Robène Isabelle, CIRAD-BIOS-UMR PVBMT (REU) - auteur correspondant
• Jouen Emmanuel, CIRAD-BIOS-UMR PVBMT (MUS) ORCID: 0009-0005-5578-023X
• Maillot-Lebon Marie-Véronique, CIRAD-BIOS-UMR PVBMT (REU)
• Fenelon Babbitha
• Hascoat Jérémy, CIRAD-BIOS-UMR PVBMT (REU)
• Pecrix Yann, CIRAD-BIOS-UMR PVBMT (REU) ORCID: 0000-0002-6537-3145
• Richard Damien
• Jaffar-Bandjee Marie-Christine, Centre hospitalier universitaire Félix Guyon (REU)
• Mavingui Patrick, Université de la Réunion (REU)
• Chiroleu Frédéric, CIRAD-BIOS-UMR PVBMT (REU) ORCID: 0000-0002-4874-5357
• Becker Nathalie, CIRAD-BIOS-UMR PVBMT (REU)
• Poubeau Patrice, Centre hospitalier universitaire Félix Guyon (REU)
• Ramiandrisoa Mahery, Laboratoires Cerballiance (REU)
• Sin Michel, Laboratoires Cerballiance (REU)
• Costet Laurent, CIRAD-BIOS-UMR PVBMT (REU) ORCID: 0000-0003-3199-2885
• Laurent Annie, Centre hospitalier universitaire Félix Guyon (REU)
• Laurent Philippe, Université de la Réunion (REU)
• Chabirand Aude, ANSES-LSV (REU)
• Moreau Aurélie, ANSES-LSV (REU)
• Reynaud Bernard ORCID: 0009-0003-6403-3944
• Jeuffrault Eric, CIRAD-DGDRD-DRRM (REU)
• Cetre-Sossah Catherine, CIRAD-BIOS-UMR ASTRE (FRA)

Source : Cirad-Agritrop (https://agritrop.cirad.fr/614088/)

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