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

Lista, Maria Jose; Matos, Pedro M.; Maguire, Thomas J. A.; Poulton, Kate; Ortiz-Zapater, Elena; Page, Robert; Sertkaya, Helin; Ortega-Prieto, Ana M.; Scourfield, Edward; O’Byrne, Aoife M.; Bouton, Clement; Dickenson, Ruth E.; Ficarelli, Mattia; Jimenez-Guardeño, Jose M.; Howard, Mark; Betancor, Gilberto; Galao, Rui Pedro; Pickering, Suzanne; Signell, Adrian W.; Wilson, Harry; Cliff, Penelope; Kia Ik, Mark Tan; Patel, Amita; MacMahon, Eithne; Cunningham, Emma; Doores, Katie; Agromayor, Monica; Martin-Serrano, Juan; Perucha, Esperanza; Mischo, Hannah E.; Shankar-Hari, Manu; Batra, Rahul; Edgeworth, Jonathan; Zuckerman, Mark; Malim, Michael H.; Neil, Stuart; Martinez-Nunez, Rocio Teresa

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/.