A two-step RT-LAMP workflow combining colorimetric screening and lateral flow assay for rapid differentiation of SARS-CoV-2 and influenza A.

Several infectious diseases share overlapping clinical symptoms and transmission routes despite being caused by distinct pathogens. Consequently, diagnostic tests capable of differentiating these infections are crucial for minimizing clinical and epidemiological misdiagnoses. However, the simultaneous detection and discrimination of multiple pathogens within a single assay remains a significant challenge, particularly in resource-limited settings. To address this, we developed a dual-readout screening strategy based on an isothermal nucleic acid amplification test (iNAAT) using pH-based colorimetric RT-LAMP (Reverse Transcription Loop-mediated Isothermal Amplification) coupled with Lateral Flow Assay (LFA). The first detection relies on a pH indicator, which confirms the presence of at least one of the target viruses in the sample. Only if the result is positive in this initial step does the assay proceed to the second detection stage, which is based on a LFA for specific identification of the virus. As a proof of concept, SARS-CoV-2 and influenza A viruses were selected as target pathogens. The duplex colorimetric RT-LAMP-LFA assay demonstrated a limit of detection of 150 RNA copies per μL for both SARS-CoV-2 and influenza A. When compared with Reverse Transcription quantitative Polymerase Chain Reaction (RT-qPCR) using a panel of real clinical samples and considering the complete two-step workflow proposed in this study, the assay achieved agreement rates of 93.33% for SARS-CoV-2, 80.00% for influenza A, and 92.59% for negative samples. Overall, the combination of dual-readout strategy improves safety and cost-effectiveness while preserving the simplicity required for point-of-care molecular diagnosis.