RUNCOV: a One-pot Triplex Real-time RT-LAMP As a Point-of-care Diagnostic Tool for Detecting SARS-CoV-2

Overview

Journal Biol Methods Protoc

Publisher Oxford University Press

Specialty Biology

Date 2025 Mar 6

PMID 40046730

Authors

Isabelle Robene

Emmanuel Jouen

Veronique Maillot-Lebon

Babbitha T Fenelon

Jeremy Hascoat

Yann Pecrix

Damien Richard

Marie-Christine Jaffar-Bandjee

Patrick Mavingui

Frederic Chiroleu

Nathalie Becker

Patrice Poubeau

Mahery Ramiandrisoa

Michel Sin

Laurent Costet

Annie Laurent

Philippe Laurent

Aude Chabirand

Aurelie Moreau

Bernard Reynaud

Eric Jeuffrault

Catherine Cetre-Sossah

Affiliations

Soon will be listed here.

Abstract

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 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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