Biotin labelled DNA-probe based detection of nucleic acid in reverse transcription-LAMP amplified oropharyngeal viral swab samples via a lateral flow assay

This study focuses on three key aspects: a) crude throat swab sample as 30 template for RT-LAMP reaction, b) biotinylated DNA probes enhanced specificity for LFA readout and c) digital semi-quantification of LFA readout. The throat swab samples from SARS-CoV-2 positive and negative patients have been used in their crude (no cleaning or pre-treatment) form for the RT-LAMP reaction. The samples were heat-inactivated but not treated for any kind of nucleic acid extraction or purification. RT-LAMP (20 min processing time) result was readout out via a LFA approach, using two labels: FITC and Biotin. FITC was enzymatically incorporated into the RT-LAMP amplicon with LF-LAMP-primer and biotin was later introduced using biotinylated DNA probes specific for the amplicon region. DNA probes based LFA readout of RT-LAMP amplicon was 97.73% sensitive and 93.10% specific. The LFA result was further analysed via a smartphone based IVD-device wherein the test line intensity was recorded. The LFA test line intensity was then correlated with the qRT-PCR CT-value of the positive swab samples. A digital semi-quantification of RT-LAMP-LFA is reported with the correlation coefficient R2= 0.7. The overall RT-LAMP-LFA assay time was recorded to be 35 min with the LoD of 4,000 RNA copies/ml.