Evaluation of the SARS-CoV-2 Inactivation Efficacy Associated With Buffers From Three Kits Used on High-Throughput RNA Extraction Platforms.

Ruth E Thom,James S Findlay,Simon A Weller,Amanda L Phelps,Karleigh A Hamblin,David O Ulaeto,Helen L Stapleton,Lin S Eastaugh,Sophie J Smither,Lyn M O’Brien

doi:10.3389/fcimb.2021.716436

Abstract

Rapid and demonstrable inactivation of SARS-CoV-2 is crucial to ensure operator safety during high-throughput testing of clinical samples. The inactivation efficacy of SARS-CoV-2 was evaluated using commercially available lysis buffers from three viral RNA extraction kits used on two high-throughput (96-well) RNA extraction platforms (Qiagen QIAcube HT and the Thermo Fisher KingFisher Flex) in combination with thermal treatment. Buffer volumes and sample ratios were chosen for their optimised suitability for RNA extraction rather than inactivation efficacy and tested against a representative sample type: SARS-CoV-2 spiked into viral transport medium (VTM). A lysis buffer mix from the MagMAX Pathogen RNA/DNA kit (Thermo Fisher), used on the KingFisher Flex, which included guanidinium isothiocyanate (GITC), a detergent, and isopropanol, demonstrated a minimum inactivation efficacy of 1 × 105 tissue culture infectious dose (TCID)50/ml. Alternative lysis buffer mixes from the MagMAX Viral/Pathogen Nucleic Acid kit (Thermo Fisher) also used on the KingFisher Flex and from the QIAamp 96 Virus QIAcube HT Kit (Qiagen) used on the QIAcube HT (both of which contained GITC and a detergent) reduced titres by 1 × 104 TCID50/ml but did not completely inactivate the virus. Heat treatment alone (15 min, 68°C) did not completely inactivate the virus, demonstrating a reduction of 1 × 103 TCID50/ml. When inactivation methods included both heat treatment and addition of lysis buffer, all methods were shown to completely inactivate SARS-CoV-2 inactivation against the viral titres tested. Results are discussed in the context of the operation of a high-throughput diagnostic laboratory.

Highlights

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the Coronaviridae family and is the causative agent of the respiratory illness, coronavirus disease 2019 (COVID-19) (Gorbalenya et al, 2020)
In this study, conducted entirely under Containment Level (CL) 3 laboratory conditions (BSL 3), we report the inactivation efficacy of SARS-CoV-2 by buffers from three commercially available kits used on these two platforms
Plates were incubated in a humidified atmosphere for 3–4 days at 37°C, after which they were scored for cytopathic effect (CPE) by microscopic observation

Summary

Introduction

Severe acute respiratory syndrome coronavirus-2 (SARS-CoV-2) belongs to the Coronaviridae family and is the causative agent of the respiratory illness, coronavirus disease 2019 (COVID-19) (Gorbalenya et al, 2020). Diagnostic testing is critical in the fight against the COVID-19 pandemic (Patel et al, 2020), not just for patients displaying symptoms and for asymptomatic carriers and presymptomatic patients (Shental et al, 2020). SARS-CoV-2 has been classified in the United Kingdom as a Hazard Group (HG) 3 pathogen by the Advisory Committee on Dangerous Pathogens (ACDP), meaning that this virus must be handled under Containment Level (CL) 3 conditions [biosafety level (BSL) 3]. Guidance from the WHO (World Health Organization 2020) and Public Health England, United Kingdom (Public Health England, 2020), has permitted non-propagative diagnostic testing to be carried out at CL 2 with non-inactivated samples being handled within a Class 1 microbiology safety cabinet

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Results

Conclusion