Amplification-Free Nucleic Acid Testing with a Fluorescence One-Step-Branched DNA-Based Lateral Flow Assay (FOB-LFA).

The emergence of the global pandemic and the discovery of nucleic acid biomarkers in cancer diagnosis have fostered the development of more accurate and adaptive molecular diagnosis technologies. Current nucleic acid testing (NAT) methods either lack sensitivity or require tedious amplification operations, which could not meet the need for point-of-care (POC) NAT for on-site and community-based diagnosis. Here, we present a fluorescence one-step-bDNA-based lateral flow assay (FOB-LFA) method for amplification-free NAT to realize point-of-care pathogen detection and disease diagnosis. Take COVID-19 as an example, the developed FOB-LFA demonstrated a high sensitivity of 300 copies/mL for the RNA of the SARS-CoV-2 pseudovirus and exhibited high specificity among various homologous pseudoviruses. Further, the result of oropharyngeal swab sample detection suggested the great potential of FOB-LFA in clinical examination. The outstanding performance of FOB-LFA, including high sensitivity, high specificity, low cost, excellent portability, and minimized risk of nucleic acid leakage and contamination, can meet the POC testing demand for the diagnosis of various infectious and genetic diseases.