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

Author

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

Subjects
Medicine, Science
Abstract

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.

Published
2021
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2. SRSF1-dependent nuclear export inhibition of C9ORF72 repeat transcripts prevents neurodegeneration and associated motor deficits

Author

Guillaume M. Hautbergue, Lydia M. Castelli, Laura Ferraiuolo, Alvaro Sanchez-Martinez, Johnathan Cooper-Knock, Adrian Higginbottom, Ya-Hui Lin, Claudia S. Bauer, Jennifer E. Dodd, Monika A. Myszczynska, Sarah M. Alam, Pierre Garneret, Jayanth S. Chandran, Evangelia Karyka, Matthew J. Stopford, Emma F. Smith, Janine Kirby, Kathrin Meyer, Brian K. Kaspar, Adrian M. Isaacs, Sherif F. El-Khamisy, Kurt J. De Vos, Ke Ning, Mimoun Azzouz, Alexander J. Whitworth, and Pamela J. Shaw

Subjects
Science
Abstract

The RNA for ALS- and frontotemporal dementia-associated C9ORF72 gene is exported from nucleus via an unknown mechanism. This study shows that reduction of nuclear export adaptor SRSF1 can alleviate neuronal cell death and nuclear export of C9ORF72 inDrosophilaand patient-derived induced motor neurons.

Published
2017
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3. Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics

Author

Laura Magro, Béatrice Jacquelin, Camille Escadafal, Pierre Garneret, Aurélia Kwasiborski, Jean-Claude Manuguerra, Fabrice Monti, Anavaj Sakuntabhai, Jessica Vanhomwegen, Pierre Lafaye, and Patrick Tabeling

Subjects
Medicine, Science
Abstract

Abstract The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

Published
2017
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4. Clinical evaluation of a RT-LAMP SARS-CoV-2 test for the Point-Of-Care, rapid, low-cost, integrating sample solid phase extraction and on which reagents are lyophilized

Author

Jean-Claude Manuguerra, Debora Freitas do Nascimento, Messaouda Merzoug, Etienne Coz, Alexandre Vilquin, Sudavip Mukherjee, Jessica Vanhomwegen, Pierre Garneret, Damien Hoinard, Jean-Christophe Olivo-Marin, Benjamin Rossi, Patrick Tabeling, elodie collin, Maxence Feher, Quentin Grassin, Elodie Brient-Litzler, and Elian Martin

Subjects
Chromatography, Chemistry, Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2), Reagent, Sample (material), Retrospective cohort study, Solid phase extraction, RNA extraction, Viral load, Point of care
Abstract

ObjectivesDetermine the sensitivity and specificity of a Point-Of-Care test (‘COVIDISC’) for SARS-COV2. The novelty of the test is to integrate, on the same (low-cost) compact plastic/paper device, solid phase RNA extraction and RT-LAMP amplification, all reagents being freeze-dried on it.MethodRetrospective study with a cohort of 99 patients characterized by real-time RT-PCR. The 37 positive naso-pharyngeal samples cover a broad range of viral loads (from 5 gc /µL to 2 106gc/ µL of sample).ResultsThe COVIDISC found 36 positives (out of 37 by IP4 RT-PCR protocols) and 63 negatives (out of 62 by RT-PCR).ConclusionThe sensitivity of the COVIDISC, found in this 99-patient retrospective study, is 97% and the specificity 100%.

Published
2021
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5. Performing point-of-care molecular testing for SARS-CoV-2 with RNA extraction and isothermal amplification

Author

Daniel Felipe Gonzalez Obando, Pierre Garneret, Vincent Enouf, Jean Claude Manuguerra, Elian Martin, Patrick Tabeling, Elodie Brient-Litzler, Sylvie van der Werf, Jean-Christophe Olivo-Marin, Jessica Vanhomwegen, Fabrice Monti, Etienne Coz, Chimie-Biologie-Innovation (UMR 8231) (CBI), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), Institut Pierre-Gilles de Gennes pour la Microfluidique, Cellule d'Intervention Biologique d'Urgence (Centre National de Référence) - Laboratory for Urgent Response to Biological Threats (National Reference Center) (CIBU), Environnement et Risques infectieux - Environment and Infectious Risks (ERI), Institut Pasteur [Paris] (IP)-Institut Pasteur [Paris] (IP), Institut Pasteur [Paris] (IP), Génétique Moléculaire des Virus à ARN - Molecular Genetics of RNA Viruses (GMV-ARN (UMR_3569 / U-Pasteur_2)), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)-Université Paris Cité (UPCité), Centre National de Référence des virus des infections respiratoires (dont la grippe) - National Reference Center Virus Influenzae [Paris] (CNR - laboratoire coordonnateur), Analyse d'images biologiques - Biological Image Analysis (BIA), Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS), Laboratoire Microfluidique, MEMS, Nanostructures (MMN), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), IPGG platform (ANR-10-EQPX-34) (PT,PG,EC,EM), Carnot Pasteur Maladies Infectieuses program (ANR 11-CARN 017-01) (JV,JCM), ANR-20-COVI-000 (EB,JV,JCM,PT,EM,C), FONDS ESPCI (EC), ANR-10-EQPX-0034,IPGG,Institut Pierre Gilles de Gennes pour la microfluidique(2010), Université Paris sciences et lettres (PSL), and The authors thank ESPCI, CNRS, IPGG platform (ANR-10-EQPX-34), PSL, PSL-VALO, PASTEUR, Carnot Pasteur Maladies Infectieuses program (ANR 11-CARN 017–01), PARIS City, ANR Flash, DIM ELICIT, Fonds de l’ESPCI, Bettencourt Fondation for their support. They thank DNA SCRIPT, France Ambassador in JAPAN, M.Ban, Bertin-Technology, Withings, T.Coulhon, JL.Missika, MC.Lemardeley, V.Croquette, A.Boquet-Pujadas, C.Batéjat, D.Hoinard, L.Dehove, D.Freitas, A.Kwasiborski, Q.Grassin, M.Feher, for discussion and help.

Subjects
RNA viruses, Viral Diseases, Time Factors, Coronaviruses, Computer science, [SDV]Life Sciences [q-bio], Artificial Gene Amplification and Extension, Polymerase Chain Reaction, Genome, Medical Conditions, COVID-19 Testing, Limit of Detection, Medicine and Health Sciences, Signal Amplification, Pathology and laboratory medicine, Virus Testing, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, Multidisciplinary, Equipment Design, Medical microbiology, Viral Load, [SDV.BBM.BP]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Biophysics, Infectious Diseases, Molecular Diagnostic Techniques, Point-of-Care Testing, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Viruses, Engineering and Technology, RNA, Viral, Medicine, RNA extraction, SARS CoV 2, Pathogens, Nucleic Acid Amplification Techniques, Research Article, SARS coronavirus, Science, Loop-mediated isothermal amplification, Computational biology, Research and Analysis Methods, Microbiology, Extraction techniques, Diagnostic Medicine, Virology, Genetics, Humans, Nucleic Acid Amplification Tests, [SPI.NANO]Engineering Sciences [physics]/Micro and nanotechnologies/Microelectronics, Molecular Biology Techniques, Molecular Biology, Point of care, Gene amplification, Biology and life sciences, SARS-CoV-2, Organisms, Viral pathogens, COVID-19, RNA, [SDV.BBM.BM]Life Sciences [q-bio]/Biochemistry, Molecular Biology/Molecular biology, Covid 19, Reverse Transcriptase-Polymerase Chain Reaction, Gold standard (test), Microbial pathogens, RNA amplification, Signal Processing, Nucleic acid, Viral Transmission and Infection
Abstract

Déposé dans medRxiv le 4 août 2020 : https://doi.org/10.1101/2020.08.01.20166538; International audience; 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.

Published
2021
Full Text
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6. Paper microfluidics for nucleic acid amplification testing (NAAT) of infectious diseases

Author

Pierre Garneret, Patrick Tabeling, Beatrice Jacquelin, Fabrice Monti, Anavaj Sakuntabhai, Pierre Lafaye, Laura Magro, Aurélia Kwasiborski, Camille Escadafal, Jessica Vanhomwegen, Jean-Claude Manuguerra, Laboratoire Microfluidique, MEMS, Nanostructures (MMN), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Centre National de la Recherche Scientifique (CNRS), Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats (CIBU), Institut Pasteur [Paris], HIV, Inflammation et persistance, Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], P. T., J. C. M. and P. L. initiated the collaboration. L. M., P. G., B. J. and C. E. prepared the manuscript with the assistance of P. T. L. M., F. M. and P. T. brought expertise on paper microfluidics and technological developments. J. C. M., A. S., C. E., A. K., J. V. and P. L. helped on more biological points of view: isothermal amplifications and sample preparations., Institut Pasteur [Paris] (IP), HIV, Inflammation et persistance - HIV, Inflammation and Persistence, and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Paper, [SDV.BIO]Life Sciences [q-bio]/Biotechnology, Microfluidics, Biomedical Engineering, Loop-mediated isothermal amplification, Bioengineering, Nanotechnology, 02 engineering and technology, Biology, 01 natural sciences, Biochemistry, Communicable Diseases, law.invention, Domain (software engineering), law, [SDV.MHEP.MI]Life Sciences [q-bio]/Human health and pathology/Infectious diseases, Lab-On-A-Chip Devices, Nucleic Acid Amplification Tests, Humans, [SDV.MHEP.ME]Life Sciences [q-bio]/Human health and pathology/Emerging diseases, 010401 analytical chemistry, General Chemistry, Nucleic acid amplification technique, Equipment Design, Lab-on-a-chip, Microfluidic Analytical Techniques, 021001 nanoscience & nanotechnology, Data science, 0104 chemical sciences, 3. Good health, Molecular Diagnostic Techniques, Low and middle income countries, Nucleic acid, 0210 nano-technology, Nucleic Acid Amplification Techniques
Abstract

International audience; The diagnosis of infectious diseases is entering a new and interesting phase. Technologies based on paper microfluidics, coupled to developments in isothermal amplification of Nucleic Acids (NAs) raise opportunities for bringing the methods of molecular biology in the field, in a low setting environment. A lot of work has been performed in the domain over the last few years and the landscape of contributions is rich and diverse. Most often, the level of sample preparation differs, along with the sample nature, the amplification and detection methods, and the design of the device, among other features. In this review, we attempt to offer a structured description of the state of the art. The domain is not mature and there exist bottlenecks that hamper the realization of Nucleic Acid Amplification Tests (NAATs) complying with the constraints of the field in low and middle income countries. In this domain however, the pace of progress is impressively fast. This review is written for a broad Lab on a Chip audience.

Published
2017
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7. Paper-based RNA detection and multiplexed analysis for Ebola virus diagnostics

Author

Camille Escadafal, Pierre Garneret, Jean-Claude Manuguerra, Anavaj Sakuntabhai, Patrick Tabeling, Pierre Lafaye, Aurélia Kwasiborski, Jessica Vanhomwegen, Fabrice Monti, Beatrice Jacquelin, Laura Magro, Gulliver (UMR 7083), Ecole Superieure de Physique et de Chimie Industrielles de la Ville de Paris (ESPCI Paris), Université Paris sciences et lettres (PSL)-Université Paris sciences et lettres (PSL)-Institut de Chimie du CNRS (INC)-Centre National de la Recherche Scientifique (CNRS), HIV, Inflammation et persistance, Institut Pasteur [Paris], Cellule d'Intervention Biologique d'Urgence - Laboratory for Urgent Response to Biological Threats (CIBU), Génétique fonctionnelle des Maladies infectieuses - Functional Genetics of Infectious Diseases, Institut Pasteur [Paris]-Centre National de la Recherche Scientifique (CNRS), Ingénierie des Anticorps (plate-forme) - Antibody Engineering (Platform), Centre National de la Recherche Scientifique (CNRS)-Institut Pasteur [Paris], The authors acknowledge financial support from the Pasteur Ebola Task Force directed by Pierre Legrain, and from Carnot Pasteur Maladies Infectieuses (ANR 11-CARN 017-01)., Sincere thanks also go to all the institutions that made the trip to Guinea possible: Pasteur Institute International Network, French and Guinean Red Crosses. For the access to the Macenta laboratory and all the administrative agreements, the authors thank Sylvain Baize, Kathleen Victoir, Chloé Pelicier, Mialy Rabenoro, Virginie Pirard, Cassandre von Platen and Nathalie Jolly. The authors are grateful to the patients, clinicians and the staff from the Ebola Treatment Center for their participation and efficient collaboration. The authors thank MicroFactory and R&DVision for the fabrication of transportable detection equipment, in a very short time., HIV, Inflammation et persistance - HIV, Inflammation and Persistence, Institut Pasteur [Paris] (IP), and Institut Pasteur [Paris] (IP)-Centre National de la Recherche Scientifique (CNRS)

Subjects
Paper, Science, [SDV]Life Sciences [q-bio], Microfluidics, Population, Recombinase Polymerase Amplification, 02 engineering and technology, Biology, medicine.disease_cause, 01 natural sciences, Multiplexing, Sensitivity and Specificity, Article, Recombinases, Lab-On-A-Chip Devices, medicine, Nucleic Acid Amplification Tests, Humans, education, [SDV.IB.BIO]Life Sciences [q-bio]/Bioengineering/Biomaterials, education.field_of_study, Multidisciplinary, Ebola virus, 010401 analytical chemistry, RNA, Reverse Transcription, Hemorrhagic Fever, Ebola, 021001 nanoscience & nanotechnology, Ebolavirus, Virology, Reverse transcriptase, 0104 chemical sciences, 3. Good health, [SDV.MP.VIR]Life Sciences [q-bio]/Microbiology and Parasitology/Virology, Medicine, Guinea, 0210 nano-technology, Biomedical engineering, Nucleic Acid Amplification Techniques, Biotechnology
Abstract

The most performing techniques enabling early diagnosis of infectious diseases rely on nucleic acid detection. Today, because of their high technicality and cost, nucleic acid amplification tests (NAAT) are of benefit only to a small fraction of developing countries population. By reducing costs, simplifying procedures and enabling multiplexing, paper microfluidics has the potential to considerably facilitate their accessibility. However, most of the studies performed in this area have not quit the lab. This letter brings NAAT on paper closer to the field, by using clinical samples and operating in a resource-limited setting. We first performed isothermal reverse transcription and Recombinase Polymerase Amplification (RT-RPA) of synthetic Ribonucleic Acid (RNA) of Ebola virus using paper microfluidics devices. We further applied this method in Guinea to detect the presence of Ebola virus in human sample RNA extracts, with minimal facilities (carry-on detection device and freeze-dried reagents on paper). RT-RPA results were available in few minutes and demonstrate a sensitivity of 90.0% compared to the gold-standard RT-PCR on a set of 43 patient samples. Furthermore, the realization of a nine-spot multilayered device achieving the parallel detection of three distinct RNA sequences opens a route toward the detection of multiple viral strains or pathogens.

Published
2017
Full Text
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