Isabelle Robène, Emmanuel Jouen, Véronique Maillot-Lebon, Babbitha T Fenelon, Jérémy Hascoat, Yann Pecrix, Damien Richard, Marie-Christine Jaffar-Bandjee, Patrick Mavingui, Frédéric Chiroleu, Nathalie Becker, Patrice Poubeau, Mahery Ramiandrisoa, Michel Sin, Laurent Costet, Annie Laurent, Philippe Laurent, Aude Chabirand, Aurélie Moreau, Bernard Reynaud, Eric Jeuffrault, Catherine Cêtre-Sossah
{"title":"RUNCOV: a one-pot triplex real-time RT-LAMP as a point-of-care diagnostic tool for detecting SARS-CoV-2.","authors":"Isabelle Robène, Emmanuel Jouen, Véronique Maillot-Lebon, Babbitha T Fenelon, Jérémy Hascoat, Yann Pecrix, Damien Richard, Marie-Christine Jaffar-Bandjee, Patrick Mavingui, Frédéric Chiroleu, Nathalie Becker, Patrice Poubeau, Mahery Ramiandrisoa, Michel Sin, Laurent Costet, Annie Laurent, Philippe Laurent, Aude Chabirand, Aurélie Moreau, Bernard Reynaud, Eric Jeuffrault, Catherine Cêtre-Sossah","doi":"10.1093/biomethods/bpaf010","DOIUrl":null,"url":null,"abstract":"<p><p>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 <i>in silico</i> 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.</p>","PeriodicalId":36528,"journal":{"name":"Biology Methods and Protocols","volume":"10 1","pages":"bpaf010"},"PeriodicalIF":2.5000,"publicationDate":"2025-02-07","publicationTypes":"Journal Article","fieldsOfStudy":null,"isOpenAccess":false,"openAccessPdf":"https://www.ncbi.nlm.nih.gov/pmc/articles/PMC11882304/pdf/","citationCount":"0","resultStr":null,"platform":"Semanticscholar","paperid":null,"PeriodicalName":"Biology Methods and Protocols","FirstCategoryId":"1085","ListUrlMain":"https://doi.org/10.1093/biomethods/bpaf010","RegionNum":0,"RegionCategory":null,"ArticlePicture":[],"TitleCN":null,"AbstractTextCN":null,"PMCID":null,"EPubDate":"2025/1/1 0:00:00","PubModel":"eCollection","JCR":"Q3","JCRName":"BIOCHEMICAL RESEARCH METHODS","Score":null,"Total":0}
引用次数: 0
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 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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