Lateral flow\u2013based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use

Saloni Agarwal,Christian Warmt,Andreas Nitsche,Frank F Bier,Livia Schrick,Joerg Henkel

doi:10.1007/s00216-022-03880-4

Saloni Agarwal, Christian Warmt + Show 4 more

Open Access

https://doi.org/10.1007/s00216-022-03880-4

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Abstract

The degree of detrimental effects inflicted on mankind by the COVID-19 pandemic increased the need to develop ASSURED (Affordable, Sensitive, Specific, User-friendly, Rapid and Robust, Equipment-free, and Deliverable) POCT (point of care testing) to overcome the current and any future pandemics. Much effort in research and development is currently advancing the progress to overcome the diagnostic pressure built up by emerging new pathogens. LAMP (loop-mediated isothermal amplification) is a well-researched isothermal technique for specific nucleic acid amplification which can be combined with a highly sensitive immunochromatographic readout via lateral flow assays (LFA). Here we discuss LAMP-LFA robustness, sensitivity, and specificity for SARS-CoV-2 N-gene detection in cDNA and clinical swab-extracted RNA samples. The LFA readout is designed to produce highly specific results by incorporation of biotin and FITC labels to 11-dUTP and LF (loop forming forward) primer, respectively. The LAMP-LFA assay was established using cDNA for N-gene with an accuracy of 95.65%. To validate the study, 82 SARS-CoV-2-positive RNA samples were tested. Reverse transcriptase (RT)-LAMP-LFA was positive for the RNA samples with an accuracy of 81.66%; SARS-CoV-2 viral RNA was detected by RT-LAMP-LFA for as low as CT-33. Our method reduced the detection time to 15 min and indicates therefore that RT-LAMP in combination with LFA represents a promising nucleic acid biosensing POCT platform that combines with smartphone based semi-quantitative data analysis. Graphical abstract

Highlights

The global COVID-19 pandemic has adversely affected humankind during the past 24 months since its first report in December 2019 in Wuhan, China [1, 2]
We present a proof of concept for an RTLAMP-lateral flow assays (LFA) for RNA of SARS-CoV-2
A bright green-yellow coloration was observed for the DNA sample and a dull orange coloration for the nontemplate control (NTC)

Summary

Introduction

The global COVID-19 pandemic has adversely affected humankind during the past 24 months since its first report in December 2019 in Wuhan, China [1, 2]. RT-PCR is efficient in confirming the presence of viral nucleic acid in the samples, but it requires time, cost of equipment and material, and trained staff to perform the PCR tests [8, 9]. These limitations urge the development of more efficient point of care testing (POCT) for the diagnostics of SARS-CoV-2 [10]. Rapid antigentest kits are being widely used, their comparatively lower sensitivity still necessitates an increase in diagnostic efficacy by exploring new approaches of nucleic acid and antigen detection, resulting in more sensitive POCT [9]

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