Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing metho...

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Autores principales:	Thomas G W Graham, Claire Dugast-Darzacq, Gina M Dailey, Xammy H Nguyenla, Erik Van Dis, Meagan N Esbin, Abrar Abidi, Sarah A Stanley, Xavier Darzacq, Robert Tjian
Formato:	article
Lenguaje:	EN
Publicado:	Public Library of Science (PLoS) 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/d2e00b68e6d249dcbc99fedaffd42153
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spelling	oai:doaj.org-article:d2e00b68e6d249dcbc99fedaffd421532021-12-02T20:05:44ZOpen-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.1932-620310.1371/journal.pone.0246647https://doaj.org/article/d2e00b68e6d249dcbc99fedaffd421532021-01-01T00:00:00Zhttps://doi.org/10.1371/journal.pone.0246647https://doaj.org/toc/1932-6203Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.Thomas G W GrahamClaire Dugast-DarzacqGina M DaileyXammy H NguyenlaErik Van DisMeagan N EsbinAbrar AbidiSarah A StanleyXavier DarzacqRobert TjianPublic Library of Science (PLoS)articleMedicineRScienceQENPLoS ONE, Vol 16, Iss 2, p e0246647 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Thomas G W Graham Claire Dugast-Darzacq Gina M Dailey Xammy H Nguyenla Erik Van Dis Meagan N Esbin Abrar Abidi Sarah A Stanley Xavier Darzacq Robert Tjian Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
description	Re-opening of communities in the midst of the ongoing COVID-19 pandemic has ignited new waves of infections in many places around the world. Mitigating the risk of reopening will require widespread SARS-CoV-2 testing, which would be greatly facilitated by simple, rapid, and inexpensive testing methods. This study evaluates several protocols for RNA extraction and RT-qPCR that are simpler and less expensive than prevailing methods. First, isopropanol precipitation is shown to provide an effective means of RNA extraction from nasopharyngeal (NP) swab samples. Second, direct addition of NP swab samples to RT-qPCRs is evaluated without an RNA extraction step. A simple, inexpensive swab collection solution suitable for direct addition is validated using contrived swab samples. Third, an open-source master mix for RT-qPCR is described that permits detection of viral RNA in NP swab samples with a limit of detection of approximately 50 RNA copies per reaction. Quantification cycle (Cq) values for purified RNA from 30 known positive clinical samples showed a strong correlation (r2 = 0.98) between this homemade master mix and commercial TaqPath master mix. Lastly, end-point fluorescence imaging is found to provide an accurate diagnostic readout without requiring a qPCR thermocycler. Adoption of these simple, open-source methods has the potential to reduce the time and expense of COVID-19 testing.
format	article
author	Thomas G W Graham Claire Dugast-Darzacq Gina M Dailey Xammy H Nguyenla Erik Van Dis Meagan N Esbin Abrar Abidi Sarah A Stanley Xavier Darzacq Robert Tjian
author_facet	Thomas G W Graham Claire Dugast-Darzacq Gina M Dailey Xammy H Nguyenla Erik Van Dis Meagan N Esbin Abrar Abidi Sarah A Stanley Xavier Darzacq Robert Tjian
author_sort	Thomas G W Graham
title	Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
title_short	Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
title_full	Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
title_fullStr	Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
title_full_unstemmed	Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.
title_sort	open-source rna extraction and rt-qpcr methods for sars-cov-2 detection.
publisher	Public Library of Science (PLoS)
publishDate	2021
url	https://doaj.org/article/d2e00b68e6d249dcbc99fedaffd42153
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Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.

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