tetano
Editor, Senior Moderator
Sci Rep
. 2021 Aug 10;11(1):16193.
doi: 10.1038/s41598-021-95607-1.
Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
Boon Lim[SUP] #[/SUP][SUP] 1 2 [/SUP], Jeremy Ratcliff[SUP] #[/SUP][SUP] 3 [/SUP], Dorota A Nawrot[SUP] #[/SUP][SUP] 4 [/SUP], Yejiong Yu[SUP] 1 2 [/SUP], Harshmeena R Sanghani[SUP] 4 [/SUP], Chia-Chen Hsu[SUP] 1 2 [/SUP], Leon Peto[SUP] 5 [/SUP], Simon Evans[SUP] 5 [/SUP], Susanne H Hodgson[SUP] 5 6 [/SUP], Aikaterini Skeva[SUP] 7 [/SUP], Maria Adam[SUP] 7 [/SUP], Maria Panopoulou[SUP] 7 [/SUP], Christos E Zois[SUP] 7 [/SUP], Katy Poncin[SUP] 8 [/SUP], Sridhar R Vasudevan[SUP] 4 [/SUP], Siqi Dai[SUP] 1 2 [/SUP], Shuai Ren[SUP] 1 2 [/SUP], Hong Chang[SUP] 9 [/SUP], Zhanfeng Cui[SUP] 1 2 9 [/SUP], Peter Simmonds[SUP] 3 [/SUP], Wei E Huang[SUP] 10 11 [/SUP], Monique I Andersson[SUP] 12 13 [/SUP]
Affiliations
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.
. 2021 Aug 10;11(1):16193.
doi: 10.1038/s41598-021-95607-1.
Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
Boon Lim[SUP] #[/SUP][SUP] 1 2 [/SUP], Jeremy Ratcliff[SUP] #[/SUP][SUP] 3 [/SUP], Dorota A Nawrot[SUP] #[/SUP][SUP] 4 [/SUP], Yejiong Yu[SUP] 1 2 [/SUP], Harshmeena R Sanghani[SUP] 4 [/SUP], Chia-Chen Hsu[SUP] 1 2 [/SUP], Leon Peto[SUP] 5 [/SUP], Simon Evans[SUP] 5 [/SUP], Susanne H Hodgson[SUP] 5 6 [/SUP], Aikaterini Skeva[SUP] 7 [/SUP], Maria Adam[SUP] 7 [/SUP], Maria Panopoulou[SUP] 7 [/SUP], Christos E Zois[SUP] 7 [/SUP], Katy Poncin[SUP] 8 [/SUP], Sridhar R Vasudevan[SUP] 4 [/SUP], Siqi Dai[SUP] 1 2 [/SUP], Shuai Ren[SUP] 1 2 [/SUP], Hong Chang[SUP] 9 [/SUP], Zhanfeng Cui[SUP] 1 2 9 [/SUP], Peter Simmonds[SUP] 3 [/SUP], Wei E Huang[SUP] 10 11 [/SUP], Monique I Andersson[SUP] 12 13 [/SUP]
Affiliations
- PMID: 34376716
- DOI: 10.1038/s41598-021-95607-1
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.