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

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

Investigation and validation of labelling loop mediated isothermal amplification (LAMP) products with different nucleotide modifications for various downstream analysis

“…Modifications of LAMP products by using dUTP instead of dTTP for usage in uracil DNA glycosylase (UDG-LAMP) assays have been shown previously 30 , 31 indicating that LAMP enzymes like Bst2.0 and Bst3.0 are able to handle this nucleotide modification without causing serious amplification problems. Only a handful of publications are known in which additional modifications like digoxigenin-dUTP (DIG-dUTP) 32 as well as fluorescent 29 or biotin labelled nucleotides 23 have been used.…”

“…2021 , we could not detect LAMP products after 10-min amplification time with our own assay. Changing the system to biotin-dUTP and FITC-labelled primer solved the problem 23 .…”

“…There is a broad range of combination possibilities of LAMP with downstream analytical systems for the detection of amplification products. They include standard gel electrophoresis 17 and quantitative amplification (qLAMP) 18 over different colorimetric reagents (phenol red 19 , leuco crystal violet 20 , SYBR green 21 , malachite green 22 ) to lateral flow assays (LFA) 23 or combinations with CRISPR-Cas Systems 24 . The most commonly used are LFA and colorimetric assays but the major limitation of the mentioned techniques is that they are not suitable for the distinction of false positive results.…”

“…Therefore, we were highly motivated to investigate the suitability of this labelling technique for other isothermal methods. Only a handful of examples exist that use modified nucleotides for direct LAMP labelling and are mostly combined with in-situ LAMP 23 , 29 . In a previous study, we demonstrated the incorporation of biotin-labeled dUTP in combination with (semi)quantitative lateral flow analysis 23 .…”

Molecularly Imprinted Polymer-Based Sensors for SARS-CoV-2: Where Are We Now?

“…Different diagnosis strategies have been developed such as RT-PCR, immunoassays (ELISA: Enzyme-Linked Immunosorbent Assay, lateral flow immunoassay), CRISPR, lateral flow-based nucleic acid detection, RT-LA, bio(mimetic)sensors, and microarray-based analysis [ 1 , 2 , 5 , 6 , 7 , 8 , 9 , 10 , 11 , 12 , 13 , 14 , 15 , 16 , 17 ]. Among the described methods, RT-PCR is the gold standard.…”