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- Title
Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection.
- Authors
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
- 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.
- Subjects
SARS-CoV-2; GENE amplification; OLIGONUCLEOTIDES; RIBONUCLEASES; COLORIMETRY
- Publication
Scientific Reports, 2021, Vol 11, Issue 1, p1
- ISSN
2045-2322
- Publication type
Article
- DOI
10.1038/s41598-021-95607-1