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Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays
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 aime...
Autores principales: | , , , , , , , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Elsevier
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878117/ https://www.ncbi.nlm.nih.gov/pubmed/33582488 http://dx.doi.org/10.1016/j.ebiom.2021.103236 |
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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. |
author_facet | 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. |
author_sort | Panpradist, Nuttada |
collection | PubMed |
description | 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 |
format | Online Article Text |
id | pubmed-7878117 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Elsevier |
record_format | MEDLINE/PubMed |
spelling | pubmed-78781172021-02-16 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. EBioMedicine Research Paper 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 Elsevier 2021-02-12 /pmc/articles/PMC7878117/ /pubmed/33582488 http://dx.doi.org/10.1016/j.ebiom.2021.103236 Text en © 2021 The Authors http://creativecommons.org/licenses/by-nc-nd/4.0/ This is an open access article under the CC BY-NC-ND license (http://creativecommons.org/licenses/by-nc-nd/4.0/). |
spellingShingle | Research Paper 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. Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title | Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title_full | Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title_fullStr | Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title_full_unstemmed | Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title_short | Simpler and faster Covid-19 testing: Strategies to streamline SARS-CoV-2 molecular assays |
title_sort | simpler and faster covid-19 testing: strategies to streamline sars-cov-2 molecular assays |
topic | Research Paper |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC7878117/ https://www.ncbi.nlm.nih.gov/pubmed/33582488 http://dx.doi.org/10.1016/j.ebiom.2021.103236 |
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