Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation.

Autor: Lista MJ; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK.; All these authors contributed equally to the completion of this work., Matos PM; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK.; All these authors contributed equally to the completion of this work., Maguire TJA; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Inflammation Biology, School of Immunology and Microbial Sciences. Asthma UK Centre in Allergic Mechanisms of Asthma. Guy's Campus, King's College London SE1 9RT, UK.; All these authors contributed equally to the completion of this work., Poulton K; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK.; All these authors contributed equally to the completion of this work., Ortiz-Zapater E; King's College London Diagnostics Team at Guy's Campus (London, UK).; Randall Centre for Cell & Molecular Biophysics. Guy's Campus, King's College London, SE1 1UL, UK.; Peter Gorer Department of Immunobiology. Guy's Campus, King's College London, SE1 9RT, UK., Page R; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Inflammation Biology, School of Immunology and Microbial Sciences. Asthma UK Centre in Allergic Mechanisms of Asthma. Guy's Campus, King's College London SE1 9RT, UK., Sertkaya H; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Ortega-Prieto AM; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., O'Byrne AM; King's College London Diagnostics Team at Guy's Campus (London, UK).; Centre for Inflammation Biology and Cancer Immunology (CIBCI). Centre for Rheumatic Diseases (CRD - EULAR Centre of Excellence). Guy's Campus, King's College London SE1 1UL, UK., Bouton C; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Dickenson RE; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Ficarelli M; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Jimenez-Guardeño JM; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Howard M; King's College London Diagnostics Team at Guy's Campus (London, UK).; Peter Gorer Department of Immunobiology. Guy's Campus, King's College London, SE1 9RT, UK., Betancor G; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Galao RP; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Pickering S; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Signell AW; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Wilson H; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Cliff P; Viapath pathology laboratories at St Thomas' Hospital., Ik MTK; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., Patel A; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., MacMahon E; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., Cunningham E; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., Doores K; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Agromayor M; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Martin-Serrano J; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Perucha E; King's College London Diagnostics Team at Guy's Campus (London, UK).; Centre for Inflammation Biology and Cancer Immunology (CIBCI). Centre for Rheumatic Diseases (CRD - EULAR Centre of Excellence). Guy's Campus, King's College London SE1 1UL, UK., Mischo HE; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Shankar-Hari M; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Batra R; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., Edgeworth J; Centre for Infectious Diseases Research, St Thomas' Hospital (London, UK)., Zuckerman M; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Virology. King's College Hospital (London, UK)., Malim MH; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Neil S; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK., Martinez-Nunez RT; King's College London Diagnostics Team at Guy's Campus (London, UK).; Dept. Infectious Diseases, School of Immunology and Microbial Sciences. Guy's Campus, King's College London SE1 9RT, UK.
Jazyk: angličtina
Zdroj: MedRxiv : the preprint server for health sciences [medRxiv] 2021 Apr 10. Date of Electronic Publication: 2021 Apr 10.
DOI: 10.1101/2020.04.22.20074351
Abstrakt: There is a worldwide need for reagents to perform SARS-CoV-2 detection. Some laboratories have implemented kit-free protocols, but many others do not have the capacity to develop these and/or perform manual processing. We provide multiple workflows for SARS-CoV-2 nucleic acid detection in clinical samples by comparing several commercially available RNA extraction methods: QIAamp Viral RNA Mini Kit (QIAgen), RNAdvance Blood/Viral (Beckman) and Mag-Bind Viral DNA/RNA 96 Kit (Omega Bio-tek). We also compared One-step RT-qPCR reagents: TaqMan Fast Virus 1-Step Master Mix (FastVirus, ThermoFisher Scientific), qPCRBIO Probe 1-Step Go Lo-ROX (PCR Biosystems) and Luna ® Universal Probe One-Step RT-qPCR Kit (Luna, NEB). We used primer-probes that detect viral N (EUA CDC) and RdRP (PHE guidelines). All RNA extraction methods provided similar results. FastVirus and Luna proved most sensitive. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrate that treatment of nasopharyngeal swabs with 70 degrees for 10 or 30 min, or 90 degrees for 10 or 30 min (both original variant and B 1.1.7) inactivates SARS-CoV-2 employing plaque assays, and that it has minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable to settings that do not have CL-3 facilities. In summary, we provide several testing pipelines that can be easily implemented in other laboratories and have made all our protocols and SOPs freely available at https://osf.io/uebvj/ .
Databáze: MEDLINE