Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA
<p dir="ltr">To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in...
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2020
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| _version_ | 1864513549637779456 |
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| author | Mohammad Rubayet Hasan (8602080) |
| author2 | Faheem Mirza (9167127) Hamad Al-Hail (9167130) Sathyavathi Sundararaju (8602086) Thabisile Xaba (9167133) Muhammad Iqbal (114924) Hashim Alhussain (9167136) Hadi Mohamad Yassine (9167139) Andres Perez-Lopez (5750300) Patrick Tang (239534) |
| author2_role | author author author author author author author author author |
| author_facet | Mohammad Rubayet Hasan (8602080) Faheem Mirza (9167127) Hamad Al-Hail (9167130) Sathyavathi Sundararaju (8602086) Thabisile Xaba (9167133) Muhammad Iqbal (114924) Hashim Alhussain (9167136) Hadi Mohamad Yassine (9167139) Andres Perez-Lopez (5750300) Patrick Tang (239534) |
| author_role | author |
| dc.creator.none.fl_str_mv | Mohammad Rubayet Hasan (8602080) Faheem Mirza (9167127) Hamad Al-Hail (9167130) Sathyavathi Sundararaju (8602086) Thabisile Xaba (9167133) Muhammad Iqbal (114924) Hashim Alhussain (9167136) Hadi Mohamad Yassine (9167139) Andres Perez-Lopez (5750300) Patrick Tang (239534) |
| dc.date.none.fl_str_mv | 2020-07-24T03:00:00Z |
| dc.identifier.none.fl_str_mv | 10.1371/journal.pone.0236564 |
| dc.relation.none.fl_str_mv | https://figshare.com/articles/journal_contribution/Detection_of_SARS-CoV-2_RNA_by_direct_RT-qPCR_on_nasopharyngeal_specimens_without_extraction_of_viral_RNA/27935283 |
| dc.rights.none.fl_str_mv | CC BY 4.0 info:eu-repo/semantics/openAccess |
| dc.subject.none.fl_str_mv | Biological sciences Microbiology Zoology SARS CoV 2 RNA extraction Virus testing COVID 19 Polymerase chain reaction Reverse transcriptase-polymerase chain reaction Heat treatment Nucleases |
| dc.title.none.fl_str_mv | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| dc.type.none.fl_str_mv | Text Journal contribution info:eu-repo/semantics/publishedVersion text contribution to journal |
| description | <p dir="ltr">To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath<sup>™</sup> 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10<sup>3</sup> copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C<sub>T</sub> values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, <i>p</i> = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.</p><h2>Other Information</h2><p dir="ltr">Published in: PLOS ONE<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1371/journal.pone.0236564" target="_blank">https://dx.doi.org/10.1371/journal.pone.0236564</a></p> |
| eu_rights_str_mv | openAccess |
| id | Manara2_0ab4c47af799d626ee366391bb124069 |
| identifier_str_mv | 10.1371/journal.pone.0236564 |
| network_acronym_str | Manara2 |
| network_name_str | Manara2 |
| oai_identifier_str | oai:figshare.com:article/27935283 |
| publishDate | 2020 |
| repository.mail.fl_str_mv | |
| repository.name.fl_str_mv | |
| repository_id_str | |
| rights_invalid_str_mv | CC BY 4.0 |
| spelling | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNAMohammad Rubayet Hasan (8602080)Faheem Mirza (9167127)Hamad Al-Hail (9167130)Sathyavathi Sundararaju (8602086)Thabisile Xaba (9167133)Muhammad Iqbal (114924)Hashim Alhussain (9167136)Hadi Mohamad Yassine (9167139)Andres Perez-Lopez (5750300)Patrick Tang (239534)Biological sciencesMicrobiologyZoologySARS CoV 2RNA extractionVirus testingCOVID 19Polymerase chain reactionReverse transcriptase-polymerase chain reactionHeat treatmentNucleases<p dir="ltr">To circumvent the limited availability of RNA extraction reagents, we aimed to develop a protocol for direct RT-qPCR to detect SARS-CoV-2 in nasopharyngeal swabs without RNA extraction. Nasopharyngeal specimens positive for SARS-CoV-2 and other coronaviruses collected in universal viral transport (UVT) medium were pre-processed by several commercial and laboratory-developed methods and tested by RT-qPCR assays without RNA extraction using different RT-qPCR master mixes. The results were compared to that of standard approach that involves RNA extraction. Incubation of specimens at 65°C for 10 minutes along with the use of TaqPath<sup>™</sup> 1-Step RT-qPCR Master Mix provides higher analytical sensitivity for detection of SARS-CoV-2 RNA than many other conditions tested. The optimized direct RT-qPCR approach demonstrated a limit of detection of 6.6x10<sup>3</sup> copy/ml and high reproducibility (co-efficient of variation = 1.2%). In 132 nasopharyngeal specimens submitted for SARS-CoV-2 testing, the sensitivity, specificity and accuracy of our optimized approach were 95%, 99% and 98.5%, respectively, with reference to the standard approach. Also, the RT-qPCR C<sub>T</sub> values obtained by the two methods were positively correlated (Pearson correlation coefficient r = 0.6971, <i>p</i> = 0.0013). The rate of PCR inhibition by the direct approach was 8% compared to 9% by the standard approach. Our simple approach to detect SARS-CoV-2 RNA by direct RT-qPCR may help laboratories continue testing for the virus despite reagent shortages or expand their testing capacity in resource limited settings.</p><h2>Other Information</h2><p dir="ltr">Published in: PLOS ONE<br>License: <a href="http://creativecommons.org/licenses/by/4.0/" target="_blank">http://creativecommons.org/licenses/by/4.0/</a><br>See article on publisher's website: <a href="https://dx.doi.org/10.1371/journal.pone.0236564" target="_blank">https://dx.doi.org/10.1371/journal.pone.0236564</a></p>2020-07-24T03:00:00ZTextJournal contributioninfo:eu-repo/semantics/publishedVersiontextcontribution to journal10.1371/journal.pone.0236564https://figshare.com/articles/journal_contribution/Detection_of_SARS-CoV-2_RNA_by_direct_RT-qPCR_on_nasopharyngeal_specimens_without_extraction_of_viral_RNA/27935283CC BY 4.0info:eu-repo/semantics/openAccessoai:figshare.com:article/279352832020-07-24T03:00:00Z |
| spellingShingle | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA Mohammad Rubayet Hasan (8602080) Biological sciences Microbiology Zoology SARS CoV 2 RNA extraction Virus testing COVID 19 Polymerase chain reaction Reverse transcriptase-polymerase chain reaction Heat treatment Nucleases |
| status_str | publishedVersion |
| title | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| title_full | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| title_fullStr | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| title_full_unstemmed | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| title_short | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| title_sort | Detection of SARS-CoV-2 RNA by direct RT-qPCR on nasopharyngeal specimens without extraction of viral RNA |
| topic | Biological sciences Microbiology Zoology SARS CoV 2 RNA extraction Virus testing COVID 19 Polymerase chain reaction Reverse transcriptase-polymerase chain reaction Heat treatment Nucleases |