Direct detection of SARS-CoV-2 using non-commercial RT-LAMP reagents on heat-inactivated samples

Alisa Alekseenko,Xiyang Liu,Simon J Elsässer,Henry Ampah-Korsah,Tomas Nyman,Rebecca J Howard,Emilia Strandback,Vicent Pelechano,Urška Rovšnik,Donal Barrett,Yerma Pareja-Sanchez,Xiushan Yin,Silvia Zuniga-Veliz,Shenglong Ye,Alexander Klenov,Gustaf Sandh,Björn Reinius,Jayshna Malloo

doi:10.1038/s41598-020-80352-8

Abstract

RT-LAMP detection of SARS-CoV-2 has been shown to be a valuable approach to scale up COVID-19 diagnostics and thus contribute to limiting the spread of the disease. Here we present the optimization of highly cost-effective in-house produced enzymes, and we benchmark their performance against commercial alternatives. We explore the compatibility between multiple DNA polymerases with high strand-displacement activity and thermostable reverse transcriptases required for RT-LAMP. We optimize reaction conditions and demonstrate their applicability using both synthetic RNA and clinical patient samples. Finally, we validate the optimized RT-LAMP assay for the detection of SARS-CoV-2 in unextracted heat-inactivated nasopharyngeal samples from 184 patients. We anticipate that optimized and affordable reagents for RT-LAMP will facilitate the expansion of SARS-CoV-2 testing globally, especially in sites and settings where the need for large scale testing cannot be met by commercial alternatives.

Highlights

The ongoing SARS-CoV-2 pandemic has had a tremendous impact on society, surpassing one million casualties worldwide and exposing the vulnerability of our globalised world to the spread of infectious disease
Displacement polymerases assayed for reverse transcriptase (RT)-LAMP polymerase activity included the Geobacillus stearothermophilus exonuclease-deficient family-A polymerase large fragment (Bst-LF), and its ‘v5.9′ and ‘v7.16′ derivatives previously reported by Ellington and colleagues to incorporate functional properties of the related Klentaq polymerase from Thermus aquaticus[20]
Addition of a thermostable RT was essential to enhance the performance of all tested enzymes, we carried out further optimizations in the presence of Superscript IV (SSIV)

Summary

Introduction

The ongoing SARS-CoV-2 pandemic has had a tremendous impact on society, surpassing one million casualties worldwide and exposing the vulnerability of our globalised world to the spread of infectious disease. Simplified isothermal approaches have the potential to facilitate SARS-CoV-2 diagnosis and contribute to the efforts against the COVID-19 pandemic This is especially important when considering that frequent testing, even at a lower sensitivity, can contribute to a decrease in spread of the d isease[1,2,3]. To make molecular testing more accessible and allow its widespread implementation, multiple approaches have been developed It has recently been shown how a simplified RT-qPCR reaction for SARS-CoV-2 detection is feasible using only one enzyme[18], or even how it is possible to use crude enzymes from lyophilized bacteria for LAMP19. We established protocols for simple production of DNA polymerases with high strand-displacement a ctivity[20] and optimized their reaction conditions for RT-LAMP We explore their compatibility with thermostable reverse transcriptases and provide a complete in-house reagent mix for fluorescent or colorimetric detection of SARS-CoV-2 RNA. We tested the ability of the produced reagents to correctly detect SARS-CoV-2 presence directly in non-purified clinical nasopharyngeal samples from 184 patients

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Conclusion