Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection

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 olig...

Descripción completa

Guardado en:

Detalles Bibliográficos
Autores principales:	Boon Lim, Jeremy Ratcliff, Dorota A. Nawrot, Yejiong Yu, Harshmeena R. Sanghani, Chia-Chen Hsu, Leon Peto, Simon Evans, Susanne H. Hodgson, Aikaterini Skeva, Maria Adam, Maria Panopoulou, Christos E. Zois, Katy Poncin, Sridhar R. Vasudevan, Siqi Dai, Shuai Ren, Hong Chang, Zhanfeng Cui, Peter Simmonds, Wei E. Huang, Monique I. Andersson
Formato:	article
Lenguaje:	EN
Publicado:	Nature Portfolio 2021
Materias:	Medicine R Science Q
Acceso en línea:	https://doaj.org/article/3a8b6fea2a544cd0a2eafc13f941fe38
Etiquetas:	Agregar Etiqueta Sin Etiquetas, Sea el primero en etiquetar este registro!

id	oai:doaj.org-article:3a8b6fea2a544cd0a2eafc13f941fe38
record_format	dspace
spelling	oai:doaj.org-article:3a8b6fea2a544cd0a2eafc13f941fe382021-12-02T19:06:40ZClinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection10.1038/s41598-021-95607-12045-2322https://doaj.org/article/3a8b6fea2a544cd0a2eafc13f941fe382021-08-01T00:00:00Zhttps://doi.org/10.1038/s41598-021-95607-1https://doaj.org/toc/2045-2322Abstract 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.Boon LimJeremy RatcliffDorota A. NawrotYejiong YuHarshmeena R. SanghaniChia-Chen HsuLeon PetoSimon EvansSusanne H. HodgsonAikaterini SkevaMaria AdamMaria PanopoulouChristos E. ZoisKaty PoncinSridhar R. VasudevanSiqi DaiShuai RenHong ChangZhanfeng CuiPeter SimmondsWei E. HuangMonique I. AnderssonNature PortfolioarticleMedicineRScienceQENScientific Reports, Vol 11, Iss 1, Pp 1-11 (2021)
institution	DOAJ
collection	DOAJ
language	EN
topic	Medicine R Science Q
spellingShingle	Medicine R Science Q Boon Lim Jeremy Ratcliff Dorota A. Nawrot Yejiong Yu Harshmeena R. Sanghani Chia-Chen Hsu Leon Peto Simon Evans Susanne H. Hodgson Aikaterini Skeva Maria Adam Maria Panopoulou Christos E. Zois Katy Poncin Sridhar R. Vasudevan Siqi Dai Shuai Ren Hong Chang Zhanfeng Cui Peter Simmonds Wei E. Huang Monique I. Andersson Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
description	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.
format	article
author	Boon Lim Jeremy Ratcliff Dorota A. Nawrot Yejiong Yu Harshmeena R. Sanghani Chia-Chen Hsu Leon Peto Simon Evans Susanne H. Hodgson Aikaterini Skeva Maria Adam Maria Panopoulou Christos E. Zois Katy Poncin Sridhar R. Vasudevan Siqi Dai Shuai Ren Hong Chang Zhanfeng Cui Peter Simmonds Wei E. Huang Monique I. Andersson
author_facet	Boon Lim Jeremy Ratcliff Dorota A. Nawrot Yejiong Yu Harshmeena R. Sanghani Chia-Chen Hsu Leon Peto Simon Evans Susanne H. Hodgson Aikaterini Skeva Maria Adam Maria Panopoulou Christos E. Zois Katy Poncin Sridhar R. Vasudevan Siqi Dai Shuai Ren Hong Chang Zhanfeng Cui Peter Simmonds Wei E. Huang Monique I. Andersson
author_sort	Boon Lim
title	Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
title_short	Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
title_full	Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
title_fullStr	Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
title_full_unstemmed	Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection
title_sort	clinical validation of optimised rt-lamp for the diagnosis of sars-cov-2 infection
publisher	Nature Portfolio
publishDate	2021
url	https://doaj.org/article/3a8b6fea2a544cd0a2eafc13f941fe38
work_keys_str_mv	AT boonlim clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT jeremyratcliff clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT dorotaanawrot clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT yejiongyu clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT harshmeenarsanghani clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT chiachenhsu clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT leonpeto clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT simonevans clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT susannehhodgson clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT aikateriniskeva clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT mariaadam clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT mariapanopoulou clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT christosezois clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT katyponcin clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT sridharrvasudevan clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT siqidai clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT shuairen clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT hongchang clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT zhanfengcui clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT petersimmonds clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT weiehuang clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection AT moniqueiandersson clinicalvalidationofoptimisedrtlampforthediagnosisofsarscov2infection
_version_	1718377142271606784

Clinical validation of optimised RT-LAMP for the diagnosis of SARS-CoV-2 infection

Ejemplares similares