Author:
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.
Abstract
AbstractWe 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.
Publisher
Springer Science and Business Media LLC
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