TY - JOUR T1 - Lateral flow–based nucleic acid detection of SARS-CoV-2 using enzymatic incorporation of biotin-labeled dUTP for POCT use AU - Agarwal, Saloni AU - Warmt, Christian AU - Henkel, Joerg AU - Schrick, Livia AU - Nitsche, Andreas AU - Bier, Frank F. AB - 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. KW - Point of care testing (POCT) KW - Lateral flow assay (LFA) KW - COVID-19 KW - Reverse transcription loop-mediated isothermal amplification (RT-LAMP) KW - SARS-CoV-2 N-gene KW - 610 Medizin und Gesundheit PY - 2022 LA - eng PB - Robert Koch-Institut JO - Analytical and Bioanalytical Chemistry VL - 414 SP - 3177 EP - 3186 DO - 10.1007/s00216-022-03880-4 ER -