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Tytuł:
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Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection.
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Autorzy:
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Lim B; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
Ratcliff J; Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, OX1 3SY, UK.
Nawrot DA; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
Yu Y; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
Sanghani HR; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
Hsu CC; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
Peto L; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.
Evans S; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.
Hodgson SH; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.; Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
Skeva A; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
Adam M; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
Panopoulou M; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
Zois CE; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
Poncin K; Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
Vasudevan SR; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
Dai S; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
Ren S; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
Chang H; Oxford Suzhou Centre for Advanced Research (OSCAR), University of Oxford, Suzhou, China.
Cui Z; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.; Oxford Suzhou Centre for Advanced Research (OSCAR), University of Oxford, Suzhou, China.
Simmonds P; Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, OX1 3SY, UK.
Huang WE; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK. .; Oxford Suzhou Centre for Advanced Research (OSCAR), University of Oxford, Suzhou, China. .
Andersson MI; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK. .; Nuffield Division of Clinical Laboratory Science, Radcliffe Department of Medicine, University of Oxford, Oxford, UK. .
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Źródło:
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Scientific reports [Sci Rep] 2021 Aug 10; Vol. 11 (1), pp. 16193. Date of Electronic Publication: 2021 Aug 10.
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Typ publikacji:
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Journal Article; Research Support, Non-U.S. Gov't; Validation Study
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Język:
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English
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Imprint Name(s):
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Original Publication: London : Nature Publishing Group, copyright 2011-
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MeSH Terms:
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COVID-19 Nucleic Acid Testing/*methods
Molecular Diagnostic Techniques/*methods
Nucleic Acid Amplification Techniques/*methods
COVID-19 Nucleic Acid Testing/standards ; Humans ; Molecular Diagnostic Techniques/standards ; Nucleic Acid Amplification Techniques/standards ; Sensitivity and Specificity
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References:
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SCR Protocol:
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LAMP assay
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Entry Date(s):
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Date Created: 20210811 Date Completed: 20210820 Latest Revision: 20210820
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Update Code:
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20240105
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PubMed Central ID:
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PMC8355225
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DOI:
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10.1038/s41598-021-95607-1
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PMID:
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34376716
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We have optimised a reverse transcription-loop-mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 from extracted RNA for clinical application. We improved the stability and reliability of the RT-LAMP assay by the addition of a temperature-dependent switch oligonucleotide to reduce self- or off-target amplification. We then developed freeze-dried master mix for single step RT-LAMP reaction, simplifying the operation for end users and improving long-term storage and transportation. The assay can detect as low as 13 copies of SARS-CoV2 RNA per reaction (25-μL). Cross reactivity with other human coronaviruses was not observed. We have applied the new RT-LAMP assay for testing clinical extracted RNA samples extracted from swabs of 72 patients in the UK and 126 samples from Greece and demonstrated the overall sensitivity of 90.2% (95% CI 83.8-94.7%) and specificity of 92.4% (95% CI 83.2-97.5%). Among 115 positive samples which Ct values were less than 34, the RT-LAMP assay was able to detect 110 of them with 95.6% sensitivity. The specificity was 100% when RNA elution used RNase-free water. The outcome of RT-LAMP can be reported by both colorimetric detection and quantifiable fluorescent reading. Objective measures with a digitized reading data flow would allow for the sharing of results for local or national surveillance.
(© 2021. The Author(s).)
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