- Record: found
- Abstract: found
- Article: found
Direct RT-qPCR detection of SARS-CoV-2 RNA from patient nasopharyngeal swabs without an RNA extraction step
research-article
Author(s):
Emily A. Bruce
1
,
* ,
Meei-Li Huang
2 ,
Garrett A. Perchetti
2 ,
Scott Tighe
3 ,
Pheobe Laaguiby
3 ,
Jessica J. Hoffman
3 ,
Diana L. Gerrard
4 ,
Arun K. Nalla
2 ,
Yulun Wei
2 ,
Alexander L. Greninger
2
,
5 ,
Sean A. Diehl
6
,
7 ,
David J. Shirley
8 ,
Debra G. B. Leonard
9
,
10 ,
Christopher D. Huston
6
,
11 ,
Beth D. Kirkpatrick
6
,
7
,
11 ,
Julie A. Dragon
3
,
6 ,
Jessica W. Crothers
9
,
10 ,
Keith R. Jerome
2
,
5
,
* ,
Jason W. Botten
1
,
6
,
7
,
*
Publication date (Electronic):
2 October 2020
Journal:
PLoS Biology
Publisher:
Public Library of Science
There is no author summary for this article yet. Authors can add summaries to their articles on ScienceOpen to make them more accessible to a non-specialist audience.
Abstract
The ongoing COVID-19 pandemic has created an unprecedented need for rapid diagnostic testing. The World Health Organization (WHO) recommends a standard assay that includes an RNA extraction step from a nasopharyngeal (NP) swab followed by reverse transcription–quantitative polymerase chain reaction (RT-qPCR) to detect the purified SARS-CoV-2 RNA. The current global shortage of RNA extraction kits has caused a severe bottleneck to COVID-19 testing. The goal of this study was to determine whether SARS-CoV-2 RNA could be detected from NP samples via a direct RT-qPCR assay that omits the RNA extraction step altogether. The direct RT-qPCR approach correctly identified 92% of a reference set of blinded NP samples ( n = 155) demonstrated to be positive for SARS-CoV-2 RNA by traditional clinical diagnostic RT-qPCR that included an RNA extraction. Importantly, the direct method had sufficient sensitivity to reliably detect those patients with viral loads that correlate with the presence of infectious virus. Thus, this strategy has the potential to ease supply choke points to substantially expand COVID-19 testing and screening capacity and should be applicable throughout the world.
Abstract
A major bottleneck to RT-PCR-based COVID-19 testing is the RNA extraction step. This study shows that a “direct RT-PCR” approach that omits the RNA extraction step can reliably detect SARS-CoV-2 RNA in nasopharyngeal swabs from individuals most likely to be infectious.
Related collections
Most cited references23
- Record: found
- Abstract: found
- Article: found
Detection of 2019 novel coronavirus (2019-nCoV) by real-time RT-PCR
Victor M Corman, Olfert Landt, Marco Kaiser … (2020)
Background The ongoing outbreak of the recently emerged novel coronavirus (2019-nCoV) poses a challenge for public health laboratories as virus isolates are unavailable while there is growing evidence that the outbreak is more widespread than initially thought, and international spread through travellers does already occur. Aim We aimed to develop and deploy robust diagnostic methodology for use in public health laboratory settings without having virus material available. Methods Here we present a validated diagnostic workflow for 2019-nCoV, its design relying on close genetic relatedness of 2019-nCoV with SARS coronavirus, making use of synthetic nucleic acid technology. Results The workflow reliably detects 2019-nCoV, and further discriminates 2019-nCoV from SARS-CoV. Through coordination between academic and public laboratories, we confirmed assay exclusivity based on 297 original clinical specimens containing a full spectrum of human respiratory viruses. Control material is made available through European Virus Archive – Global (EVAg), a European Union infrastructure project. Conclusion The present study demonstrates the enormous response capacity achieved through coordination of academic and public laboratories in national and European research networks.
- Record: found
- Abstract: found
- Article: not found
Virological assessment of hospitalized patients with COVID-2019
Roman Wölfel, Victor M Corman, Wolfgang Guggemos … (2020)
Coronavirus disease 2019 (COVID-19) is an acute infection of the respiratory tract that emerged in late 20191,2. Initial outbreaks in China involved 13.8% of cases with severe courses, and 6.1% of cases with critical courses3. This severe presentation may result from the virus using a virus receptor that is expressed predominantly in the lung2,4; the same receptor tropism is thought to have determined the pathogenicity-but also aided in the control-of severe acute respiratory syndrome (SARS) in 20035. However, there are reports of cases of COVID-19 in which the patient shows mild upper respiratory tract symptoms, which suggests the potential for pre- or oligosymptomatic transmission6-8. There is an urgent need for information on virus replication, immunity and infectivity in specific sites of the body. Here we report a detailed virological analysis of nine cases of COVID-19 that provides proof of active virus replication in tissues of the upper respiratory tract. Pharyngeal virus shedding was very high during the first week of symptoms, with a peak at 7.11 × 108 RNA copies per throat swab on day 4. Infectious virus was readily isolated from samples derived from the throat or lung, but not from stool samples-in spite of high concentrations of virus RNA. Blood and urine samples never yielded virus. Active replication in the throat was confirmed by the presence of viral replicative RNA intermediates in the throat samples. We consistently detected sequence-distinct virus populations in throat and lung samples from one patient, proving independent replication. The shedding of viral RNA from sputum outlasted the end of symptoms. Seroconversion occurred after 7 days in 50% of patients (and by day 14 in all patients), but was not followed by a rapid decline in viral load. COVID-19 can present as a mild illness of the upper respiratory tract. The confirmation of active virus replication in the upper respiratory tract has implications for the containment of COVID-19.
- Record: found
- Abstract: found
- Article: not found
Presymptomatic SARS-CoV-2 Infections and Transmission in a Skilled Nursing Facility
Melissa M. Arons, Kelly Hatfield, Sujan Reddy … (2020)
Abstract Background Severe acute respiratory syndrome coronavirus 2 (SARS-CoV-2) infection can spread rapidly within skilled nursing facilities. After identification of a case of Covid-19 in a skilled nursing facility, we assessed transmission and evaluated the adequacy of symptom-based screening to identify infections in residents. Methods We conducted two serial point-prevalence surveys, 1 week apart, in which assenting residents of the facility underwent nasopharyngeal and oropharyngeal testing for SARS-CoV-2, including real-time reverse-transcriptase polymerase chain reaction (rRT-PCR), viral culture, and sequencing. Symptoms that had been present during the preceding 14 days were recorded. Asymptomatic residents who tested positive were reassessed 7 days later. Residents with SARS-CoV-2 infection were categorized as symptomatic with typical symptoms (fever, cough, or shortness of breath), symptomatic with only atypical symptoms, presymptomatic, or asymptomatic. Results Twenty-three days after the first positive test result in a resident at this skilled nursing facility, 57 of 89 residents (64%) tested positive for SARS-CoV-2. Among 76 residents who participated in point-prevalence surveys, 48 (63%) tested positive. Of these 48 residents, 27 (56%) were asymptomatic at the time of testing; 24 subsequently developed symptoms (median time to onset, 4 days). Samples from these 24 presymptomatic residents had a median rRT-PCR cycle threshold value of 23.1, and viable virus was recovered from 17 residents. As of April 3, of the 57 residents with SARS-CoV-2 infection, 11 had been hospitalized (3 in the intensive care unit) and 15 had died (mortality, 26%). Of the 34 residents whose specimens were sequenced, 27 (79%) had sequences that fit into two clusters with a difference of one nucleotide. Conclusions Rapid and widespread transmission of SARS-CoV-2 was demonstrated in this skilled nursing facility. More than half of residents with positive test results were asymptomatic at the time of testing and most likely contributed to transmission. Infection-control strategies focused solely on symptomatic residents were not sufficient to prevent transmission after SARS-CoV-2 introduction into this facility.
Author and article information
Contributors
Emily A. Bruce:
ORCID: http://orcid.org/0000-0001-8391-370X
Role: ConceptualizationRole: Data curationRole: Formal analysisRole: MethodologyRole:
Writing – original draftRole: Writing – review & editing
Meei-Li Huang: Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole:
MethodologyRole: Writing – review & editing
Garrett A. Perchetti:
ORCID: http://orcid.org/0000-0003-3786-0509
Role: InvestigationRole: MethodologyRole: Writing – review & editing
Scott Tighe:
ORCID: http://orcid.org/0000-0002-3988-0741
Role: ConceptualizationRole: Data curationRole: Formal analysisRole: InvestigationRole:
MethodologyRole: Writing – review & editing
Pheobe Laaguiby: Role: Data curationRole: InvestigationRole: MethodologyRole: Writing – review & editing
Jessica J. Hoffman:
ORCID: http://orcid.org/0000-0002-9286-1611
Role: Data curationRole: InvestigationRole: MethodologyRole: Writing – review & editing
Diana L. Gerrard: Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole:
Writing – review & editing
Arun K. Nalla: Role: InvestigationRole: MethodologyRole: Writing – review & editing
Yulun Wei: Role: Data curationRole: Formal analysisRole: InvestigationRole: MethodologyRole:
Writing – review & editing
Alexander L. Greninger:
ORCID: http://orcid.org/0000-0002-7443-0527
Role: ResourcesRole: Writing – review & editing
Sean A. Diehl:
ORCID: http://orcid.org/0000-0001-9700-235X
Role: Writing – review & editing
David J. Shirley:
ORCID: http://orcid.org/0000-0003-4529-0403
Role: Data curationRole: Formal analysisRole: Writing – review & editing
Debra G. B. Leonard:
ORCID: http://orcid.org/0000-0003-1431-4935
Role: Writing – review & editing
Christopher D. Huston:
ORCID: http://orcid.org/0000-0002-5393-139X
Role: Writing – original draftRole: Writing – review & editing
Beth D. Kirkpatrick: Role: Writing – review & editing
Julie A. Dragon: Role: Funding acquisitionRole: ResourcesRole: Writing – review & editing
Jessica W. Crothers:
ORCID: http://orcid.org/0000-0003-4594-5051
Role: InvestigationRole: ResourcesRole: Writing – review & editing
Keith R. Jerome:
ORCID: http://orcid.org/0000-0002-8212-3789
Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole:
InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing
– original draftRole: Writing – review & editing
Jason W. Botten:
ORCID: http://orcid.org/0000-0001-6839-0306
Role: ConceptualizationRole: Data curationRole: Formal analysisRole: Funding acquisitionRole:
InvestigationRole: Project administrationRole: ResourcesRole: SupervisionRole: Writing
– original draftRole: Writing – review & editing
Raquel L. Lieberman: Role: Academic Editor
Journal
Journal ID (nlm-ta): PLoS Biol
Journal ID (iso-abbrev): PLoS Biol
Journal ID (publisher-id): plos
Journal ID (pmc): plosbiol
Title:
PLoS Biology
Publisher:
Public Library of Science
(San Francisco, CA USA
)
ISSN
(Print):
1544-9173
ISSN
(Electronic):
1545-7885
Publication date
(Electronic):
2
October
2020
Publication date Collection:
October
2020
Publication date PMC-release: 2
October
2020
Volume: 18
Issue: 10
Electronic Location Identifier: e3000896
Affiliations
[1
]
Division of Immunobiology, Department of Medicine, Robert Larner, M.D. College of
Medicine, University of Vermont, Burlington, Vermont, United States of America
[2
]
Virology Division, Department of Laboratory Medicine and Pathology, University of
Washington, Seattle, Washington, United States of America
[3
]
Vermont Integrative Genomics Resource, Robert Larner, M.D. College of Medicine, University
of Vermont, Burlington, Vermont, United States of America
[4
]
Department of Pathology and Laboratory Medicine, University of Vermont Medical Center,
Burlington, Vermont, United States of America
[5
]
Vaccine and Infectious Disease Division, Fred Hutchinson Cancer Research Center, Seattle,
Washington, United States of America
[6
]
Department of Microbiology and Molecular Genetics, Robert Larner, M.D. College of
Medicine, University of Vermont, Burlington, Vermont, United States of America
[7
]
Vaccine Testing Center, Robert Larner, M.D. College of Medicine, University of Vermont,
Burlington, Vermont, United States of America
[9
]
Department of Pathology and Laboratory Medicine, Robert Larner, M.D. College of Medicine,
University of Vermont, Burlington, Vermont, United States of America
Author notes
I have read the journal's policy and the authors of this manuscript have no competing interests. While DJS was employed at IXIS LLC at the time of this study, his employment there did not create a competing interest. Further, IXIS LLC had no involvement in this study.
* E-mail:
emily.bruce@
123456med.uvm.edu
(EAB);
kjerome@
123456fredhutch.org
(KRJ);
jason.botten@
123456med.uvm.edu
(JWB)
Author information
Emily A. Bruce
http://orcid.org/0000-0001-8391-370X
Garrett A. Perchetti
http://orcid.org/0000-0003-3786-0509
Scott Tighe
http://orcid.org/0000-0002-3988-0741
Jessica J. Hoffman
http://orcid.org/0000-0002-9286-1611
Alexander L. Greninger
http://orcid.org/0000-0002-7443-0527
Sean A. Diehl
http://orcid.org/0000-0001-9700-235X
David J. Shirley
http://orcid.org/0000-0003-4529-0403
Debra G. B. Leonard
http://orcid.org/0000-0003-1431-4935
Christopher D. Huston
http://orcid.org/0000-0002-5393-139X
Jessica W. Crothers
http://orcid.org/0000-0003-4594-5051
Keith R. Jerome
http://orcid.org/0000-0002-8212-3789
Jason W. Botten
http://orcid.org/0000-0001-6839-0306
Article
Publisher ID: PBIOLOGY-D-20-01258DOI: 10.1371/journal.pbio.3000896
PMC ID: 7556528
PubMed ID: 33006983
SO-VID: c607576b-b7ab-4e43-9253-27f81dc1150c
Copyright © © 2020 Bruce et al
License:
This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.
History
Date
received
: 4
May
2020
Date
accepted
: 10
September
2020
Page count
Figures: 3,
Tables: 4,
Pages: 14
Funding
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P30GM118228-04
Award Recipient
:
Julie A. Dragon
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P20GM125498-02
Award Recipient
:
Beth D. Kirkpatrick
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: U01AI1141997
Award Recipient
:
Jason W. Botten
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: R41AI132047
Award Recipient
:
Jason W. Botten
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: U54 GM115516-01
Award Recipient
:
Julie A. Dragon
Funded by: University of Vermont
Award ID: Office of the Vice President for Research at the University of Vermont
Award Recipient
:
Jason W. Botten
Funded by: funder-id http://dx.doi.org/10.13039/100000937, M.J. Murdock Charitable Trust;
Award Recipient
:
ORCID: http://orcid.org/0000-0002-8212-3789
Keith R. Jerome
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P30GM118228-04
Award Recipient
:
ORCID: http://orcid.org/0000-0001-8391-370X
Emily A. Bruce
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P20GM125498-02
Award Recipient
:
ORCID: http://orcid.org/0000-0002-5393-139X
Christopher D. Huston
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P20GM125498-02
Award Recipient
:
ORCID: http://orcid.org/0000-0001-9700-235X
Sean A. Diehl
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: P20GM125498-02
Award Recipient
:
Julie A. Dragon
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: U01AI1141997
Award Recipient
:
ORCID: http://orcid.org/0000-0001-9700-235X
Sean A. Diehl
Funded by: funder-id http://dx.doi.org/10.13039/100000002, National Institutes of Health;
Award ID: U01AI1141997
Award Recipient
:
Beth D. Kirkpatrick
The authors gratefully acknowledge funding from the Office of the Vice President for
Research at the University of Vermont (JWB), the M.J. Murdock Charitable Trust (KRJ),
and National Institute of Health (NIH) grants P30GM118228-04 (Immunology and Infectious
Disease COBRE) (EB, JAD), P20GM125498-02 (Translational Global Infectious Diseases
Research COBRE) (BDK, CDH, SAD, JAD), U01AI1141997 (JWB, SAD, and BDK), R41AI132047
(JWB), and U54 GM115516-01 (JAD). Funding agencies had no role in study design, collection
and analysis of data, manuscript preparation or the decision to publish.
Categories
Subject:
Methods and Resources
Subject:
Research and analysis methods
Subject:
Extraction techniques
Subject:
RNA extraction
Subject:
Physical Sciences
Subject:
Materials Science
Subject:
Materials
Subject:
Mixtures
Subject:
Solutions
Subject:
Diluents
Subject:
Biology and life sciences
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Viruses
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RNA viruses
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Coronaviruses
Subject:
SARS coronavirus
Subject:
SARS CoV 2
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Biology and life sciences
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Microbiology
Subject:
Medical microbiology
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Microbial pathogens
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Viral pathogens
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Coronaviruses
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SARS coronavirus
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Subject:
Medicine and health sciences
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Pathology and laboratory medicine
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Pathogens
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Microbial pathogens
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Coronaviruses
Subject:
SARS coronavirus
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SARS CoV 2
Subject:
Biology and life sciences
Subject:
Organisms
Subject:
Viruses
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Viral pathogens
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Coronaviruses
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SARS coronavirus
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SARS CoV 2
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Medicine and Health Sciences
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Diagnostic Medicine
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Infectious Diseases
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Subject:
Covid 19
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Biology and Life Sciences
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Molecular Biology
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Molecular Biology Techniques
Subject:
Artificial Gene Amplification and Extension
Subject:
Polymerase Chain Reaction
Subject:
Reverse Transcriptase-Polymerase Chain Reaction
Subject:
Research and Analysis Methods
Subject:
Molecular Biology Techniques
Subject:
Artificial Gene Amplification and Extension
Subject:
Polymerase Chain Reaction
Subject:
Reverse Transcriptase-Polymerase Chain Reaction
Subject:
Biology and life sciences
Subject:
Biochemistry
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Proteins
Subject:
DNA-binding proteins
Subject:
Nucleases
Subject:
Ribonucleases
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Biology and Life Sciences
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Biochemistry
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Enzymology
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Enzymes
Subject:
Hydrolases
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Nucleases
Subject:
Ribonucleases
Subject:
Biology and Life Sciences
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Biochemistry
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Proteins
Subject:
Enzymes
Subject:
Hydrolases
Subject:
Nucleases
Subject:
Ribonucleases
Subject:
Biology and Life Sciences
Subject:
Molecular Biology
Subject:
Molecular Biology Techniques
Subject:
Artificial Gene Amplification and Extension
Subject:
Polymerase Chain Reaction
Subject:
Research and Analysis Methods
Subject:
Molecular Biology Techniques
Subject:
Artificial Gene Amplification and Extension
Subject:
Polymerase Chain Reaction
Custom metadata
PLOS Publication Stage vor-update-to-uncorrected-proof
Publication Update 2020-10-14
Data Availability The authors declare that all data supporting the findings of this study are available
within the paper and its Supporting information files.
Outbreaks COVID-19
ScienceOpen disciplines: Life sciences
Data availability:
ScienceOpen disciplines: Life sciences