RT-RAA with a lateral flow assay readout based on ssDNA hybridization for detection of RNA viruses – the case of SARS-CoV-2

The conjugation of Reverse-Transcription Recombinase-Aided Amplification (RT-RAA) and Nucleic Acid Lateral Flow Assays (NALFAs) is a useful strategy for the point-of-care detection of viruses like SARS-CoV-2. In this work we developed an end-point diagnostic method combining RT-RAA and NALFA that is based on the hybridization of ssDNA and relies exclusively on the interaction of nucleic acids for producing a specific signal.Different tails were used to modify a previously published set of primers targeting the RdRp gene, enabling the test of distinct sequences and lengths in terms of amplification efficiency. Additionally, RT-RAA amplification conditions, including the temperature and time of reaction, were optimized. Lateral flow strips were assembled and running conditions were adjusted. The best combination of tailed primers was selected and the resulting amplicons were successfully detected in the test strip. The developed RT-RAA-NALFA method showed a limit of detection (LOD) of ~12 RNA copies/µL and no cross-reactivity was seen testing the DNA/RNA of 16 different pathogens except for other SARS-CoV species, thus exhibiting good analytical sensitivity and specificity. The blind test of 50 clinical samples by RT-RAA-NALFA, showed a high agreement (18/21 positives and 28/29 negatives were correctly identified) with real-time RT-PCR results. These results lead to an overall clinical sensitivity of ~86% and an overall clinical specificity of ~97%. The clinical sensitivity value increases to ~94% for viral loads corresponding to a quantification cycle (Cq) below 30 in real-time RT-PCR. This emphasizes the suitability of the test for the efficient detection of RNA virus like SARS-CoV-2.