Abstract PO-075: Performance comparison of five extraction kits for SARS-CoV-2 RNA extraction
Authors: Lungu Ilinca M. , De Luca Angela , Li Jason , Bayani Jane , Spears Melanie , Pugh Trevor J. , Bartlett John M.S. 
Published: December 2020

Abstract
A new type of coronavirus, SARS-CoV-2, was identified in January 2020. Its associated disease, COVID-19, was announced as a pandemic by the World Health Organization in March 2020. The Ontario Institute for Cancer Research quickly engaged to support viral sequencing, not only in frontline health care workers but in cancer patients. A key deliverable was the selection of an extraction methodology that would not impact the supply of approved diagnostic testing reagents. This consideration was in response to reports of possible shortages predicted early in the pandemic and as indicated by the Public Health Agency of Canada (PHAC), through their call for reagents in April 2020. Five commercially available kits for automated nucleic acid extraction were compared. The KingFisher Flex Purification System (ThermoFisher, 5400610) was used for nucleic acid extraction. Four kits were selected 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 Acid Isolation 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 MagMAX Viral/Pathogen Kit (MVP; ThermoFisher, A42352), approved by the Food and Drug Administration of Canada for diagnostic 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 demonstrated reproducible yields, while yields from the Total RNA kit were inconsistent. The CORE and Omega kits possessed the best overall extraction efficiencies (both 70%). The MVP kit and Total NA kit were 59% and 44% efficient in recovery, respectively. The CORE and Omega kits ranked best after overall assessment. Patient samples were subsequently extracted using both kits and successfully sequenced. Extraction kits do not all perform to the same specification. In our hands, we found the MVP kit did not perform as well as others, despite being approved for diagnostic use, and the Total RNA kit showed inconsistent results. Many reagents are commercially available and should be explored as alternatives to the approved SARS-CoV-2 diagnostic reagents, particularly during a global crisis. Interestingly, following our validation testing, supply of the CORE kit became limited with unknown future availability. This illustrated the need to validate multiple methods during uncertain times in order to maintain critical testing.
Citation Format: Ilinca M. Lungu, Angela De Luca, Jason Li, Jane Bayani, Melanie Spears, Trevor J. Pugh, John M.S. Bartlett. Performance comparison of five extraction kits for SARS-CoV-2 RNA extraction [abstract]. In: Proceedings of the AACR Virtual Meeting: COVID-19 and Cancer; 2020 Jul 20-22. Philadelphia (PA): AACR; Clin Cancer Res 2020;26(18_Suppl):Abstract nr PO-075.