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

2021; Public Library of Science; Volume: 16; Issue: 9 Linguagem: Inglês

10.1371/journal.pone.0256813

1932-6203

María José Lista, Pedro M. Matos, Thomas J. A. Maguire, Kate Poulton, Elena Ortiz‐Zapater, Robert L. Page, Helin Sertkaya, Ana María Ortega-Prieto, Edward J. Scourfield, Aoife M. O’Byrne, Clément R. Bouton, Ruth E. Dickenson, Mattia Ficarelli, Jose M. Jiménez-Guardeño, Mark Howard, Gilberto Betancor, Rui Pedro Galão, Suzanne Pickering, Adrian W. Signell, Harry Wilson, Penelope R. Cliff, Mark Tan Kia Ik, Amita Patel, Eithne MacMahon, Emma Cunningham, Katie J. Doores, Monica Agromayor, Juan Martin‐Serrano, Esperanza Perucha, Hannah E. Mischo, Manu Shankar‐Hari, Rahul Batra, Jonathan Edgeworth, Mark Zuckerman, Michael H. Malim, Stuart J. D. Neil, Rocío T. Martínez-Nuñez,

Biosensors and Analytical Detection

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/ .