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Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs
The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection o...
Autores principales: | , , , , , , |
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Formato: | Online Artículo Texto |
Lenguaje: | English |
Publicado: |
Public Library of Science
2021
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Materias: | |
Acceso en línea: | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8448339/ https://www.ncbi.nlm.nih.gov/pubmed/34534259 http://dx.doi.org/10.1371/journal.pone.0257563 |
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author | Jones, Les Bakre, Abhijeet Naikare, Hemant Kolhe, Ravindra Sanchez, Susan Mosley, Yung-Yi C. Tripp, Ralph A. |
author_facet | Jones, Les Bakre, Abhijeet Naikare, Hemant Kolhe, Ravindra Sanchez, Susan Mosley, Yung-Yi C. Tripp, Ralph A. |
author_sort | Jones, Les |
collection | PubMed |
description | The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity. |
format | Online Article Text |
id | pubmed-8448339 |
institution | National Center for Biotechnology Information |
language | English |
publishDate | 2021 |
publisher | Public Library of Science |
record_format | MEDLINE/PubMed |
spelling | pubmed-84483392021-09-18 Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs Jones, Les Bakre, Abhijeet Naikare, Hemant Kolhe, Ravindra Sanchez, Susan Mosley, Yung-Yi C. Tripp, Ralph A. PLoS One Research Article The COVID-19 pandemic caused by the SARS-CoV-2 is a serious health threat causing worldwide morbidity and mortality. Real-time reverse transcription PCR (RT-qPCR) is currently the standard for SARS-CoV-2 detection. Although various nucleic acid-based assays have been developed to aid the detection of SARS-CoV-2 from COVID-19 patient samples, the objective of this study was to develop a diagnostic test that can be completed in 30 minutes without having to isolate RNA from the samples. Here, we present an RNA amplification detection method performed using reverse transcription loop-mediated isothermal amplification (RT-LAMP) reactions to achieve specific, rapid (30 min), and sensitive (<100 copies) fluorescent detection in real-time of SARS-CoV-2 directly from patient nasopharyngeal swab (NP) samples. When compared to RT-qPCR, positive NP swab samples assayed by fluorescent RT-LAMP had 98% (n = 41/42) concordance and negative NP swab samples assayed by fluorescent RT-LAMP had 87% (n = 59/68) concordance for the same samples. Importantly, the fluorescent RT-LAMP results were obtained without purification of RNA from the NP swab samples in contrast to RT-qPCR. We also show that the fluorescent RT-LAMP assay can specifically detect live virus directly from cultures of both SARS-CoV-2 wild type (WA1/2020), and a SARS-CoV-2 B.1.1.7 (alpha) variant strain with equal sensitivity to RT-qPCR. RT-LAMP has several advantages over RT-qPCR including isothermal amplification, speed (<30 min), reduced costs, and similar sensitivity and specificity. Public Library of Science 2021-09-17 /pmc/articles/PMC8448339/ /pubmed/34534259 http://dx.doi.org/10.1371/journal.pone.0257563 Text en © 2021 Jones et al https://creativecommons.org/licenses/by/4.0/This is an open access article distributed under the terms of the Creative Commons Attribution License (https://creativecommons.org/licenses/by/4.0/) , which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited. |
spellingShingle | Research Article Jones, Les Bakre, Abhijeet Naikare, Hemant Kolhe, Ravindra Sanchez, Susan Mosley, Yung-Yi C. Tripp, Ralph A. Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title | Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title_full | Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title_fullStr | Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title_full_unstemmed | Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title_short | Isothermal amplification and fluorescent detection of SARS-CoV-2 and SARS-CoV-2 variant virus in nasopharyngeal swabs |
title_sort | isothermal amplification and fluorescent detection of sars-cov-2 and sars-cov-2 variant virus in nasopharyngeal swabs |
topic | Research Article |
url | https://www.ncbi.nlm.nih.gov/pmc/articles/PMC8448339/ https://www.ncbi.nlm.nih.gov/pubmed/34534259 http://dx.doi.org/10.1371/journal.pone.0257563 |
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