Performance comparison of fiveextraction kits for SARS-CoV-2 RNA extraction

ABSTRACT

A new type of coronavirus, SARS-CoV-2, was identified in January 2020. Its associated disease, COVID-19, wasannounced as a pandemic by the World Health Organization in March 2020. The Ontario Institute for CancerResearch quickly engaged to support viral sequencing, not only in frontline health care workers but in cancerpatients. A key deliverable was the selection of an extraction methodology that would not impact the supply ofapproved diagnostic testing reagents. This consideration was in response to reports of possible shortages predictedearly in the pandemic and as indicated by the Public Health Agency of Canada (PHAC), through their call forreagents in April 2020. Five commercially available kits for automated nucleic acid extraction were compared. TheKingFisher Flex Purification System (ThermoFisher, 5400610) was used for nucleic acid extraction. Four kits wereselected based on availability, system compatibility, and exclusion from PHAC's call for COVID-19 testing reagents.The MagMAX CORE Nucleic Acid Purification Kit (CORE;ThermoFisher, A32702), MagMAX Total Nucleic AcidIsolation Kit (Total NA;ThermoFisher, AM1840), MagMAX Total RNA Isolation Kit (Total RNA;ThermoFisher, AM1830), and Mag-Bind Viral DNA/RNA 96 Kit (Omega;Omega BioTek, M6246-03) were evaluated. The MagMAXViral/Pathogen Kit (MVP;ThermoFisher, A42352), approved by the Food and Drug Administration of Canada fordiagnostic testing, was used as a benchmark. Test samples were prepared using Universal Human RNA (Agilent,740000), lambda DNA solution (Sigma Aldrich, ERMAD442K), SARS-CoV-2 RNA (ATCC, VR1986D) and heat-inactivated virus (ATCC, VR-1986HK). Extractions were performed by two operators on replicate samples. Protocols were assessed on reproducibility, yield, reagent availability, run time, and ease of use. The top two kits were validated with nasopharyngeal swab samples from SARS-CoV-2-positive patients. Four of five kits demonstratedreproducible yields, while yields from the Total RNA kit were inconsistent. The CORE and Omega kits possessedthe best overall extraction efficiencies (both 70%). The MVP kit and Total NA kit were 59% and 44% efficient inrecovery, respectively. The CORE and Omega kits ranked best after overall assessment. Patient samples weresubsequently extracted using both kits and successfully sequenced. Extraction kits do not all perform to the samespecification. In our hands, we found the MVP kit did not perform as well as others, despite being approved fordiagnostic use, and the Total RNA kit showed inconsistent results. Many reagents are commercially available andshould be explored as alternatives to the approved SARS-CoV-2 diagnostic reagents, particularly during a globalcrisis. Interestingly, following our validation testing, supply of the CORE kit became limited with unknown futureavailability. This illustrated the need to validate multiple methods during uncertain times in order to maintain criticaltesting.