TY - JOUR
T1 - Resilient SARS-CoV-2 diagnostics workflows including viral heat inactivation
AU - Lista, Maria Jose
AU - Matos, Pedro M
AU - Maguire, Thomas J A
AU - Poulton, Kate
AU - Ortiz-Zapater, Elena
AU - Page, Robert
AU - Sertkaya, Helin
AU - Ortega-Prieto, Ana M
AU - Scourfield, Edward
AU - O'Byrne, Aoife M
AU - Bouton, Clement
AU - Dickenson, Ruth E
AU - Ficarelli, Mattia
AU - Jimenez-Guardeño, Jose M
AU - Howard, Mark
AU - Betancor, Gilberto
AU - Galao, Rui Pedro
AU - Pickering, Suzanne
AU - Signell, Adrian W
AU - Wilson, Harry
AU - Cliff, Penelope
AU - Kia Ik, Mark Tan
AU - Patel, Amita
AU - MacMahon, Eithne
AU - Cunningham, Emma
AU - Doores, Katie
AU - Agromayor, Monica
AU - Martin-Serrano, Juan
AU - Perucha, Esperanza
AU - Mischo, Hannah E
AU - Shankar-Hari, Manu
AU - Batra, Rahul
AU - Edgeworth, Jonathan
AU - Zuckerman, Mark
AU - Malim, Michael H
AU - Neil, Stuart
AU - Martinez-Nunez, Rocio Teresa
N1 - Publisher Copyright:
Copyright: © 2021 Lista et al. This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
PY - 2021/9/15
Y1 - 2021/9/15
N2 - 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. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in 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/.
AB - 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. RNA extraction methods provided similar results, with Beckman performing better with our primer-probe combinations. Luna proved most sensitive although overall the three reagents did not show significant differences. N detection was more reliable than that of RdRP, particularly in samples with low viral titres. Importantly, we demonstrated that heat treatment of nasopharyngeal swabs at 70°C for 10 or 30 min, or 90°C for 10 or 30 min (both original variant and B 1.1.7) inactivated SARS-CoV-2 employing plaque assays, and had minimal impact on the sensitivity of the qPCR in clinical samples. These findings make SARS-CoV-2 testing portable in 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/.
KW - COVID-19/diagnosis
KW - COVID-19 Testing/methods
KW - Epidemics/prevention & control
KW - Hot Temperature
KW - Humans
KW - Nasopharynx/virology
KW - RNA, Viral/genetics
KW - Reagent Kits, Diagnostic
KW - Reproducibility of Results
KW - Reverse Transcriptase Polymerase Chain Reaction/methods
KW - SARS-CoV-2/genetics
KW - Sensitivity and Specificity
KW - Specimen Handling/methods
KW - Virus Inactivation
KW - Workflow
UR - http://www.scopus.com/inward/record.url?scp=85115028416&partnerID=8YFLogxK
U2 - 10.1371/journal.pone.0256813
DO - 10.1371/journal.pone.0256813
M3 - Article
C2 - 34525109
SN - 1932-6203
VL - 16
JO - PLoS ONE
JF - PLoS ONE
IS - 9 September
M1 - e0256813
ER -