Joel D Pearson1,2,3, Daniel Trcka1, Suying Lu1,2,3, Sharon J Hyduk4, Mark Jen1,5, Marie-Ming Aynaud1, J Javier Hernández1,6, Philippos Peidis1,2,3, Miriam Barrios-Rodiles1,5, Kin Chan1, Jim Woodgett1,7, Tony Mazzulli3,8, Liliana Attisano9, Laurence Pelletier1,6, Myron I Cybulsky3,4, Jeffrey L Wrana1,5,6, Rod Bremner10,11,12. 1. Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada. 2. Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Canada. 3. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. 4. Toronto General Hospital Research Institute, University Health Network, Toronto, Canada. 5. Network Collaborative Biology Centre, Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Toronto, Canada. 6. Department of Molecular Genetics, University of Toronto, Toronto, Canada. 7. Department of Medical Biophysics, University of Toronto, Toronto, Canada. 8. Department of Microbiology, Sinai Health System/University Health Network, Toronto, Canada. 9. Department of Biochemistry, Donnelly Centre, University of Toronto, Toronto, Canada. 10. Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada. bremner@lunenfeld.ca. 11. Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Canada. bremner@lunenfeld.ca. 12. Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada. bremner@lunenfeld.ca.
Abstract
BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.
BACKGROUND: Sensitive, rapid, and accessible diagnostics continue to be critical to track the COVID-19 pandemic caused by the SARS-CoV-2 virus. RT-qPCR is the gold standard test, and comparison of methodologies and reagents, utilizing patient samples, is important to establish reliable diagnostic pipelines. METHODS: Here, we assessed indirect methods that require RNA extraction with direct RT-qPCR on patient samples. Four different RNA extraction kits (Qiagen, Invitrogen, BGI and Norgen Biotek) were compared. For detection, we assessed two recently developed Taqman-based modules (BGI and Norgen Biotek), a SYBR green-based approach (NEB Luna Universal One-Step Kit) with published and newly-developed primers, and clinical results (Seegene STARMag RNA extraction system and Allplex 2019-nCoV RT-qPCR assay). We also tested and optimized direct, extraction-free detection using these RT-qPCR systems and performed a cost analysis of the different methods evaluated here. RESULTS: Most RNA isolation procedures performed similarly, and while all RT-qPCR modules effectively detected purified viral RNA, the BGI system provided overall superior performance (lower detection limit, lower Ct values and higher sensitivity), generating comparable results to original clinical diagnostic data, and identifying samples ranging from 65 copies to 2.1 × 105 copies of viral genome/μl. However, the BGI detection system is more expensive than other options tested here. With direct RT-qPCR, simply adding an RNase inhibitor greatly improved detection, without the need for any other treatments (e.g. lysis buffers or boiling). The best direct methods detected ~ 10 fold less virus than indirect methods, but this simplified approach reduced sample handling, as well as assay time and cost. CONCLUSIONS: With extracted RNA, the BGI RT-qPCR detection system exhibited superior performance over the Norgen system, matching initial clinical diagnosis with the Seegene Allplex assay. The BGI system was also suitable for direct, extraction-free analysis, providing 78.4% sensitivity. The Norgen system, however, still accurately detected samples with a clinical Ct < 33 from extracted RNA, provided significant cost savings, and was superior to SYBR green assays that exhibited reduced specificity.
Entities:
Keywords:
COVID-19; Direct detection; RT-qPCR; SARS-CoV-2
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