Journal Article

Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

van der Werf, Sylvie, Monti, Fabrice, Enouf, Vincent, Martin, Elian, González Obando, Daniel Felipe, Olivo-Marin, Jean-Christophe, Garneret, Pierre, Tabeling, Patrick, Vanhomwegen, Jessica, Brient-Litzler, Elodie, Manuguerra, Jean-Claude, Coz, Etienne

2021

Overview

To respond to the urgent need for COVID-19 testing, countries perform nucleic acid amplification tests (NAAT) for the detection of SARS-CoV-2 in centralized laboratories. Real-time RT—PCR (Reverse transcription—Polymerase Chain Reaction), used to amplify and detect the viral RNA., is considered, as the current gold standard for diagnostics. It is an efficient process, but the complex engineering required for automated RNA extraction and temperature cycling makes it incompatible for use in point of care settings [1]. In the present work, by harnessing progress made in the past two decades in isothermal amplification and paper microfluidics, we created a portable test, in which SARS-CoV-2 RNA is extracted, amplified isothermally by RT—LAMP (Loop-mediated Isothermal Amplification), and detected using intercalating dyes or fluorescent probes. Depending on the viral load in the tested samples, the detection takes between twenty minutes and one hour. Using a set of 16 pools of naso-pharyngal swab eluates, we estimated a limit of detection comparable to real-time RT-PCR (i.e. 1 genome copies per microliter of clinical sample) and no cross‐reaction with eight major respiratory viruses currently circulating in Europe. We designed and fabricated an easy-to-use portable device called “COVIDISC” to carry out the test at the point of care. The low cost of the materials along with the absence of complex equipment will expedite the widespread dissemination of this device. What is proposed here is a new efficient tool to help managing the pandemics.

• Publisher: Public Library of Science,Public Library of Science (PLoS)
• Subject: Acids / Amplification / Biology and life sciences / Coronaviruses / COVID-19 / COVID-19 - diagnosis / COVID-19 Testing - economics / COVID-19 Testing - instrumentation / Diagnostic systems / Disks / Editing / Engineering and Technology / Equipment Design / Ethanol / Evaluation / Genomes / Humans / Influenza / Influenza A / Influenza B / Laboratories / Life Sciences / Limit of Detection / Medicine and Health Sciences / Membranes / Methodology / Molecular diagnostic techniques / Molecular Diagnostic Techniques - economics / Molecular Diagnostic Techniques - instrumentation / Nucleic Acid Amplification Techniques - economics / Nucleic Acid Amplification Techniques - instrumentation / Nucleic acids / Pathogens / Point of care testing / Point-of-Care Testing - economics / Polymerase chain reaction / Research and Analysis Methods / Respiratory diseases / Respiratory syncytial virus / Reverse transcription / Rhinovirus / Ribonucleic acid / RNA / RNA, Viral - genetics / RNA, Viral - isolation & purification / SARS-CoV-2 - genetics / SARS-CoV-2 - isolation & purification / Severe acute respiratory syndrome / Severe acute respiratory syndrome coronavirus 2 / Time Factors / Viral diseases / Viruses