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Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection.
Lim, Boon; Ratcliff, Jeremy; Nawrot, Dorota A; Yu, Yejiong; Sanghani, Harshmeena R; Hsu, Chia-Chen; Peto, Leon; Evans, Simon; Hodgson, Susanne H; Skeva, Aikaterini; Adam, Maria; Panopoulou, Maria; Zois, Christos E; Poncin, Katy; Vasudevan, Sridhar R; Dai, Siqi; Ren, Shuai; Chang, Hong; Cui, Zhanfeng; Simmonds, Peter; Huang, Wei E; Andersson, Monique I.
  • Lim B; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.
  • Ratcliff J; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
  • Nawrot DA; Peter Medawar Building for Pathogen Research, Nuffield Department of Medicine, University of Oxford, Oxford, OX1 3SY, UK.
  • Yu Y; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
  • Sanghani HR; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.
  • Hsu CC; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
  • Peto L; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
  • Evans S; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.
  • Hodgson SH; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
  • Skeva A; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.
  • Adam M; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.
  • Panopoulou M; Department of Microbiology, Oxford University NHS Foundation Trust, Oxford, UK.
  • Zois CE; Jenner Institute, Nuffield Department of Medicine, University of Oxford, Oxford, UK.
  • Poncin K; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
  • Vasudevan SR; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
  • Dai S; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
  • Ren S; Democritus University of Thrace, University Hospital of Alexandroupolis, Alexandroupoli, Greece.
  • Chang H; Sir William Dunn School of Pathology, University of Oxford, Oxford, UK.
  • Cui Z; Department of Pharmacology, University of Oxford, Mansfield Road, Oxford, UK.
  • Simmonds P; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.
  • Huang WE; Department of Engineering Science, Institute of Biomedical Engineering, University of Oxford, Oxford, OX3 7DQ, UK.
  • Andersson MI; Department of Engineering Science, University of Oxford, Parks Road, Oxford, OX1 3PJ, UK.
Sci Rep ; 11(1): 16193, 2021 08 10.
Article in English | MEDLINE | ID: covidwho-1351975
ABSTRACT
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.
Subject(s)

Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acid Amplification Techniques / Molecular Diagnostic Techniques / COVID-19 Nucleic Acid Testing Type of study: Diagnostic study / Randomized controlled trials Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-95607-1

Full text: Available Collection: International databases Database: MEDLINE Main subject: Nucleic Acid Amplification Techniques / Molecular Diagnostic Techniques / COVID-19 Nucleic Acid Testing Type of study: Diagnostic study / Randomized controlled trials Limits: Humans Language: English Journal: Sci Rep Year: 2021 Document Type: Article Affiliation country: S41598-021-95607-1