A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR

Research output: Contribution to journalJournal articleResearchpeer-review

Documents

  • Fulltext

    Final published version, 499 KB, PDF document

The SARS-CoV-2 pandemic has created an urgent need for diagnostic tests to detect viral RNA. Commercial RNA extraction kits are often expensive, in limited supply, and do not always fully inactivate the virus. Together, this calls for the development of safer methods for SARS-CoV-2 extraction that utilize readily available reagents and equipment present in most standard laboratories. We optimized and simplified a RNA extraction method combining a high molar acidic guanidinium isothiocyanate (GITC) solution, phenol and chloroform. First, we determined the GITC/RNA dilution thresholds compatible with an efficient two-step RT-qPCR for B2M mRNA in nasopharyngeal (NP) or oropharyngeal (OP) swab samples. Second, we optimized a one-step RT-qPCR against SARS-CoV-2 using NP and OP samples. We furthermore tested a SARS-CoV-2 dilution series to determine the detection threshold. The method enables downstream detection of SARS-CoV-2 by RT-qPCR with high sensitivity (~4 viral RNA copies per RT-qPCR). The protocol is simple, safe, and expands analysis capacity as the inactivated samples can be used in RT-qPCR detection tests at laboratories not otherwise classified for viral work. The method takes about 30 min from swab to PCR-ready viral RNA and circumvents the need for commercial RNA purification kits.

Original languageEnglish
JournalAPMIS
Volume129
Issue number7
Pages (from-to)393-400
Number of pages8
ISSN0903-4641
DOIs
Publication statusPublished - 2021

Bibliographical note

Publisher Copyright:
© 2021 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology

    Research areas

  • clinical microbiology, molecular microbiology, rapid diagnostic methods, RNA extraction, RT-qPCR, SARS-CoV-2, SARS-CoV-2 RT-qPCR, virology, virus inactivation

ID: 302569642