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

TY - UNPB

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 - 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 - Ik, Mark Tan Kia

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

PY - 2021/4/10

Y1 - 2021/4/10

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 (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/ .

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 (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/ .

U2 - 10.1101/2020.04.22.20074351

DO - 10.1101/2020.04.22.20074351

M3 - Preprint

C2 - 33851184

T3 - MedRxiv

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

ER -