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Comparison of SARS-CoV-2 indirect and direct RT-qPCR detection methods.
Pearson, Joel D; Trcka, Daniel; Lu, Suying; Hyduk, Sharon J; Jen, Mark; Aynaud, Marie-Ming; Hernández, J Javier; Peidis, Philippos; Barrios-Rodiles, Miriam; Chan, Kin; Woodgett, Jim; Mazzulli, Tony; Attisano, Liliana; Pelletier, Laurence; Cybulsky, Myron I; Wrana, Jeffrey L; Bremner, Rod.
  • Pearson JD; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Trcka D; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Canada.
  • Lu S; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
  • Hyduk SJ; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Jen M; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Aynaud MM; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Canada.
  • Hernández JJ; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
  • Peidis P; Toronto General Hospital Research Institute, University Health Network, Toronto, Canada.
  • Barrios-Rodiles M; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Chan K; Network Collaborative Biology Centre, Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Toronto, Canada.
  • Woodgett J; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Mazzulli T; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Attisano L; Department of Molecular Genetics, University of Toronto, Toronto, Canada.
  • Pelletier L; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
  • Cybulsky MI; Department of Ophthalmology and Vision Science, University of Toronto, Toronto, Canada.
  • Wrana JL; Department of Laboratory Medicine and Pathobiology, University of Toronto, Toronto, Canada.
  • Bremner R; Lunenfeld-Tanenbaum Research Institute, Mt Sinai Hospital, Sinai Health System, Toronto, Canada.
Virol J ; 18(1): 99, 2021 05 17.
Article in English | MEDLINE | ID: covidwho-1232429
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.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reagent Kits, Diagnostic / Specimen Handling / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Limits: Humans Language: English Journal: Virol J Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: S12985-021-01574-4

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Full text: Available Collection: International databases Database: MEDLINE Main subject: Reagent Kits, Diagnostic / Specimen Handling / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Limits: Humans Language: English Journal: Virol J Journal subject: Virology Year: 2021 Document Type: Article Affiliation country: S12985-021-01574-4