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

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14 Citations (Scopus)

Abstract

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/.
Original languageEnglish
Article numbere0256813
JournalPLoS ONE
Volume16
Issue number9 September
DOIs
Publication statusPublished - 15 Sept 2021

Keywords

  • COVID-19/diagnosis
  • COVID-19 Testing/methods
  • Epidemics/prevention & control
  • Hot Temperature
  • Humans
  • Nasopharynx/virology
  • RNA, Viral/genetics
  • Reagent Kits, Diagnostic
  • Reproducibility of Results
  • Reverse Transcriptase Polymerase Chain Reaction/methods
  • SARS-CoV-2/genetics
  • Sensitivity and Specificity
  • Specimen Handling/methods
  • Virus Inactivation
  • Workflow

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