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

Boon Huat Lim ,Dorota A Nawrot,Wei E Huang,Chia-Chen Hsu,Susanne H Hodgson,Hong Chang,Μαρία Αδάμ ,Aikaterini Skeva,Peter Simmonds,Maria Panopoulou,Christos E Zois,Katy Poncin,Sridhar Vasudevan ,Susan E Evans ,Shuai Ren,Zhanfeng Cui,Leon Peto,Siqi Dai,Jeremy Ratcliff,Yejiong Yu,Harshmeena Sanghani ,Monique Andersson

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.

Highlights

The rapid spread of SARS-CoV-2 has challenged the testing capacity of many countries
To address the problem of self- and off-target amplification, we introduced a short oligonucleotide designated as a switch, whose sequence is complementary to one of the primers used in the LAMP reaction and 3′-end was modified by a dark quencher molecule—Iowa Black RQ (IBRQ)
A working hypothesis for the mechanism of action is that when the temperature is below the working temperature of LAMP (e.g. 65 °C), the FIP primer is bound to the switch oligonucleotide

Summary

Introduction

The rapid spread of SARS-CoV-2 has challenged the testing capacity of many countries. We propose a reverse transcription, loop mediated isothermal amplification (RT-LAMP) assay for the detection of SARS-CoV-2 RNA. By shortening the sample collection, testing, and readout, the POCT RT-LAMP assay is suited to settings where real-time results would directly impact on patient care, including mobile testing centres, emergency departments, primary care facilities, residential homes, and a irports[5]. There, we demonstrated the ability of our O117 primer set to reliably detect 20 copies per reaction of an Orf1ab RNA transcript. In this project, we have extended on our previous formulation in several ways to further streamline and stabilise the performance of this assay as a POCT outside of standard diagnostic laboratories. We validate the new formulation using several commercial SARS-CoV-2 transcripts, residual oropharyngeal clinical samples and saliva clinical samples

Methods

Results

Conclusion