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...
Main Authors: | , , , , , , , , , |
---|---|
Format: | Article |
Language: | English |
Published: |
Public Library of Science (PLoS)
2021-01-01
|
Series: | PLoS ONE |
Online Access: | https://doi.org/10.1371/journal.pone.0246647 |
id |
doaj-d2e00b68e6d249dcbc99fedaffd42153 |
---|---|
record_format |
Article |
spelling |
doaj-d2e00b68e6d249dcbc99fedaffd421532021-03-04T13:05:46ZengPublic Library of Science (PLoS)PLoS ONE1932-62032021-01-01162e024664710.1371/journal.pone.0246647Open-source RNA extraction and RT-qPCR methods for SARS-CoV-2 detection.Thomas G W GrahamClaire Dugast-DarzacqGina M DaileyXammy H NguyenlaErik Van DisMeagan N EsbinAbrar AbidiSarah A StanleyXavier DarzacqRobert TjianRe-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.https://doi.org/10.1371/journal.pone.0246647 |
collection |
DOAJ |
language |
English |
format |
Article |
sources |
DOAJ |
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 |
spellingShingle |
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. PLoS ONE |
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) |
series |
PLoS ONE |
issn |
1932-6203 |
publishDate |
2021-01-01 |
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. |
url |
https://doi.org/10.1371/journal.pone.0246647 |
work_keys_str_mv |
AT thomasgwgraham opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT clairedugastdarzacq opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT ginamdailey opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT xammyhnguyenla opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT erikvandis opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT meagannesbin opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT abrarabidi opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT sarahastanley opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT xavierdarzacq opensourcernaextractionandrtqpcrmethodsforsarscov2detection AT roberttjian opensourcernaextractionandrtqpcrmethodsforsarscov2detection |
_version_ |
1714800774452609024 |