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RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWAB USING QIAGEN RNEASY KITS OR DIRECTLY VIA OMISSION OF AN RNA EXTRACTION STEP

View ORCID ProfileEmily A. Bruce, Scott Tighe, Jessica J. Hoffman, Pheobe Laaguiby, Diana L. Gerrard, View ORCID ProfileSean A. Diehl, View ORCID ProfileDebra G. B. Leonard, Christopher D. Huston, Beth D. Kirkpatrick, Jessica W. Crothers, View ORCID ProfileJulie Dragon, View ORCID ProfileJason Botten
doi: https://doi.org/10.1101/2020.03.20.001008
Emily A. Bruce
1Department of Medicine, Division of Immmunobiology, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
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  • ORCID record for Emily A. Bruce
  • For correspondence: Emily.bruce@med.uvm.edu Jason.botten@med.uvm.edu
Scott Tighe
2Vermont Integrative Genomics Resource, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
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Jessica J. Hoffman
2Vermont Integrative Genomics Resource, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
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Pheobe Laaguiby
2Vermont Integrative Genomics Resource, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
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Diana L. Gerrard
3Department of Pathology and Laboratory Medicine, University of Vermont Medical Center, Burlington VT, 05401, USA
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Sean A. Diehl
4Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
5Vaccine Testing Center, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, VT, 05405 USA
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Debra G. B. Leonard
6Department of Pathology and Laboratory Medicine, Robert Larner, M.D. College of Medicine, University of Vermont and the University of Vermont Health Network, Burlington VT, 05405, USA
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Christopher D. Huston
4Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
7Department of Medicine, Division of Infectious Disease, University of Vermont Medical Center, Burlington VT, 05401, USA
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Beth D. Kirkpatrick
4Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
5Vaccine Testing Center, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, VT, 05405 USA
7Department of Medicine, Division of Infectious Disease, University of Vermont Medical Center, Burlington VT, 05401, USA
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Jessica W. Crothers
6Department of Pathology and Laboratory Medicine, Robert Larner, M.D. College of Medicine, University of Vermont and the University of Vermont Health Network, Burlington VT, 05405, USA
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Julie Dragon
2Vermont Integrative Genomics Resource, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
4Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
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Jason Botten
1Department of Medicine, Division of Immmunobiology, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
4Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington VT, 05405, USA
5Vaccine Testing Center, Robert Larner, M.D. College of Medicine, University of Vermont, Burlington, VT, 05405 USA
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  • For correspondence: Emily.bruce@med.uvm.edu Jason.botten@med.uvm.edu
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ABSTRACT

The ongoing SARS-CoV-2 pandemic has caused an unprecedented need for rapid diagnostic screening [1]. The primary assay employed is a reverse transcription quantitative polymerase chain reaction (RT-qPCR) assay that requires the use of an RNA extraction kit [2,3]. In the United States, the Centers for Disease Control and Prevention (CDC) protocol requires the use of the Qiagen QIAamp DSP Viral RNA Mini kit. The current shortage of this RNA extraction kit during this pandemic has resulted in a severe bottleneck in testing capacity. To address this problem, we tested two alternative strategies: the use of alternative RNA extraction kits or a direct RT-qPCR assay that omits an RNA extraction step altogether. We found that the Qiagen RNeasy Mini kit and the Qiagen RNeasy Micro kit could be substituted for the QIAamp Viral RNA Mini kit. Importantly, we report here that the RT-qPCR assay can be performed directly on patient sample material from a nasal swab, without the need for an RNA extraction step of any kind. Collectively, our findings provide viable options to circumvent supply chain issues in COVID-19 testing. Further, the ability to omit the RNA extraction step from RT-qPCR screening protocols would drastically ease supply chokepoints of COVID-19 screening and should be applicable throughout the world. We would note that our findings are preliminary and based on a single pooled nasopharyngeal swab sample from two previously confirmed positive COVID-19 patients. But due to the urgent need for high volume COVID-19 screening, we wanted to make these findings available immediately while we conduct replicate studies using additional patient samples.

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The copyright holder for this preprint is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under a CC-BY-NC-ND 4.0 International license.
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Posted March 21, 2020.
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RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWAB USING QIAGEN RNEASY KITS OR DIRECTLY VIA OMISSION OF AN RNA EXTRACTION STEP
Emily A. Bruce, Scott Tighe, Jessica J. Hoffman, Pheobe Laaguiby, Diana L. Gerrard, Sean A. Diehl, Debra G. B. Leonard, Christopher D. Huston, Beth D. Kirkpatrick, Jessica W. Crothers, Julie Dragon, Jason Botten
bioRxiv 2020.03.20.001008; doi: https://doi.org/10.1101/2020.03.20.001008
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RT-qPCR DETECTION OF SARS-CoV-2 RNA FROM PATIENT NASOPHARYNGEAL SWAB USING QIAGEN RNEASY KITS OR DIRECTLY VIA OMISSION OF AN RNA EXTRACTION STEP
Emily A. Bruce, Scott Tighe, Jessica J. Hoffman, Pheobe Laaguiby, Diana L. Gerrard, Sean A. Diehl, Debra G. B. Leonard, Christopher D. Huston, Beth D. Kirkpatrick, Jessica W. Crothers, Julie Dragon, Jason Botten
bioRxiv 2020.03.20.001008; doi: https://doi.org/10.1101/2020.03.20.001008

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