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

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.

Keywords: PCR; SARS-CoV-2; biosafety; clinical diagnosis; high throughput; laboratory-acquired infection.

Figure 1

Titre of SARS-CoV-2 by TCID₅₀ assay following inactivation protocols. (A) Qiagen protocol (Virus QIAcube HT Kit). (B) MagMAX Protocol 1 (Pathogen DNA/RNA). (C) MagMAX Protocol 2 (Viral Pathogen Kit). Geometric Mean + Geometric Standard Deviation collated from triplicate results from three separate occasions (n = 9). Dashed line = lower limit of quantification (LLoQ < 32 TCID₅₀/ml); tissue culture medium (TCM); <LLoQ on graph indicates viable virus was recovered in some replicates but was below limit of quantification. Kruskal–Wallis ANOVA with Dunn’s multiple comparison post hoc, where ***p < 0.001, ****p < 0.0001; statistical analysis excludes virus stock and lysis only data.