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

Lelde Kalnina,Jan Gerstoft,Àngels Mateu‐Regué,Annemette Hald,Henrik Oerum,Finn Cilius Nielsen,Stephanie Oerum,Astrid K N Iversen

doi:10.1111/apm.13123

Abstract

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.

Highlights

France, have large numbers of COVID-19 patients [6]
To investigate how guanidinium isothiocyanate (GITC) affected the efficiency of each step of an reverse-transcriptase quantitative polymerase chain reaction (RT-qPCR) reaction, we set up twostep B2M RT-qPCRs in the presence of variating concentrations of GITC
The ability to test large segments of the population represents the most effective means of managing the SARS-CoV-2 pandemic and making informed decisions on the reopening of our societies. To date, such tests typically combine the use of a front-end RNA extraction kit and a one-step RT-qPCR detection kit

Summary

Introduction

France, have large numbers of COVID-19 patients [6]. As of today, cases of COVID-19 are rapidly increasing in India, Mexico and parts of Africa and South America. The effect of GITC on a corresponding one-step SARSCoV-2 RT-qPCR reaction was examined using the dilution series of extracted RNA from OP and NP samples from the COVID-19 patient.

Results

Conclusion