TY - JOUR

T1 - A simple, safe and sensitive method for SARS-CoV-2 inactivation and RNA extraction for RT-qPCR

AU - Kalnina, Lelde

AU - Mateu-Regué, Àngels

AU - Oerum, Stephanie

AU - Hald, Annemette

AU - Gerstoft, Jan

AU - Oerum, Henrik

AU - Nielsen, Finn Cilius

AU - Iversen, Astrid K.N.

N1 - Publisher Copyright: © 2021 The Authors. APMIS published by John Wiley & Sons Ltd on behalf of Scandinavian Societies for Medical Microbiology and Pathology

PY - 2021

Y1 - 2021

N2 - 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.

AB - 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.

KW - clinical microbiology

KW - molecular microbiology

KW - rapid diagnostic methods

KW - RNA extraction

KW - RT-qPCR

KW - SARS-CoV-2

KW - SARS-CoV-2 RT-qPCR

KW - virology

KW - virus inactivation

U2 - 10.1111/apm.13123

DO - 10.1111/apm.13123

M3 - Journal article

C2 - 33730407

AN - SCOPUS:85102593074

VL - 129

SP - 393

EP - 400

JO - A P M I S. Acta Pathologica, Microbiologica et Immunologica Scandinavica

JF - A P M I S. Acta Pathologica, Microbiologica et Immunologica Scandinavica

SN - 0903-4641

IS - 7

ER -