Author: 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.
Title: Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays Cord-id: 3adn8xd1 Document date: 2021_2_12
ID: 3adn8xd1
Snippet: 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:
Document: 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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