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Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays.
Panpradist, Nuttada; Wang, Qin; Ruth, Parker S; Kotnik, Jack H; Oreskovic, Amy K; Miller, Abraham; Stewart, Samuel W A; Vrana, Justin; Han, Peter D; Beck, Ingrid A; Starita, Lea M; Frenkel, Lisa M; Lutz, Barry R.
  • Panpradist N; Department of Bioengineering, University of Washington, Seattle, WA, United States; Global Health of Women, Adolescents, and Children (Global WACh), School of Public Health, University of Washington, Seattle, WA, United States.
  • Wang Q; Department of Bioengineering, University of Washington, Seattle, WA, United States.
  • Ruth PS; Department of Bioengineering, University of Washington, Seattle, WA, United States; Paul G. Allen School of Computer Science & Engineering, University of Washington, Seattle, WA, United States.
  • Kotnik JH; Department of Bioengineering, University of Washington, Seattle, WA, United States; Department of Family Medicine, University of Washington, Seattle, WA, United States.
  • Oreskovic AK; Department of Bioengineering, University of Washington, Seattle, WA, United States.
  • Miller A; Department of Bioengineering, University of Washington, Seattle, WA, United States.
  • Stewart SWA; Department of Bioengineering, University of Washington, Seattle, WA, United States; Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA, United States.
  • Vrana J; Department of Bioengineering, University of Washington, Seattle, WA, United States.
  • Han PD; Department of Genome Sciences, Seattle, WA, United States; Brotman Baty Institute for Precision Medicine, Seattle, WA, United States.
  • Beck IA; Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA, United States.
  • Starita LM; Department of Genome Sciences, Seattle, WA, United States; Brotman Baty Institute for Precision Medicine, Seattle, WA, United States.
  • Frenkel LM; Center for Global Infectious Disease Research, Seattle Children's Research Institute, Seattle, WA, United States; Departments of Global Health, Medicine, Paediatrics, and Laboratory Medicine, University of Washington, Seattle, WA, United States. Electronic address: lfrenkel@uw.edu.
  • Lutz BR; Department of Bioengineering, University of Washington, Seattle, WA, United States; Brotman Baty Institute for Precision Medicine, Seattle, WA, United States. Electronic address: blutz@uw.edu.
EBioMedicine ; 64: 103236, 2021 Feb.
Article in English | MEDLINE | ID: covidwho-1083288
ABSTRACT

BACKGROUND:

Detection of SARS-CoV-2 infections is important for treatment, isolation of infected and exposed individuals, and contact tracing. RT-qPCR is the "gold-standard" method to sensitively detect SARS-CoV-2 RNA, but most laboratory-developed RT-qPCR assays involve complex steps. Here, we aimed to simplify RT-qPCR assays by streamlining reaction setup, eliminating RNA extraction, and proposing reduced-cost detection workflows that avoid the need for expensive qPCR instruments.

METHOD:

A low-cost RT-PCR based "kit" was developed for faster turnaround than the CDC developed protocol. We demonstrated three detection workflows two that can be deployed in laboratories conducting assays of variable complexity, and one that could be simple enough for point-of-care. Analytical sensitivity was assessed using SARS-CoV-2 RNA spiked in simulated nasal matrix. Clinical performance was evaluated using contrived human nasal matrix (n = 41) and clinical nasal specimens collected from individuals with respiratory symptoms (n = 110).

FINDING:

The analytical sensitivity of the lyophilised RT-PCR was 10 copies/reaction using purified SARS-CoV-2 RNA, and 20 copies/reaction when using direct lysate in simulated nasal matrix. Evaluation of assay performance on contrived human matrix showed 96.7-100% specificity and 100% sensitivity at ≥20 RNA copies. A head-to-head comparison with the standard CDC protocol on clinical specimens showed 83.8-94.6% sensitivity and 96.8-100% specificity. We found 3.6% indeterminate samples (undetected human control), lower than 8.1% with the standard protocol.

INTERPRETATION:

This preliminary work should support laboratories or commercial entities to develop and expand access to Covid-19 testing. Software guidance development for this assay is ongoing to enable implementation in other settings. FUND USA NIH R01AI140845 and Seattle Children's Research Institute.
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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / Reverse Transcriptase Polymerase Chain Reaction / Real-Time Polymerase Chain Reaction / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Limits: Humans Language: English Journal: EBioMedicine Year: 2021 Document Type: Article Affiliation country: J.ebiom.2021.103236

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Full text: Available Collection: International databases Database: MEDLINE Main subject: RNA, Viral / Reverse Transcriptase Polymerase Chain Reaction / Real-Time Polymerase Chain Reaction / COVID-19 Nucleic Acid Testing / SARS-CoV-2 / COVID-19 Type of study: Diagnostic study / Experimental Studies / Prognostic study Limits: Humans Language: English Journal: EBioMedicine Year: 2021 Document Type: Article Affiliation country: J.ebiom.2021.103236