Pre-amplified dsDNA-Tag lateral flow assay for highly sensitive point-of-care detection of influenza A H1N1

The current immediate detection of influenza A (H1N1) virus faces the dual challenges of insufficient sensitivity and the risk of amplification contamination. Here, we developed a novel lateral flow nucleic acid biosensor using pre-amplified dsDNA tag (PADT). Linearized M13 phage was preamplified to generate a 3.5-kbp dsDNA product containing a 20-nt overhang probe and heavy biotin tags, followed by the formation of FAM probe/target RNA/PADT complexes by a sandwich-type detection strategy. RESULT: This technique achieves a 1 pM detection limit for H1N1-specific RNA, demonstrating 100-fold greater sensitivity than conventional lateral flow chromatography (p < 0.01). This method eliminates nucleic acid amplification, preventing false positives from amplicon contamination. This method effectively prevents false positives induced by product contamination by avoiding direct amplification of the target nucleic acid. This stepwise strategy, marrying pre-amplification with point-of-need detection, achieves laboratory-grade accuracy while maintaining field applicability. The step-by-step strategy of pre-amplification and immediate detection combines laboratory precision with field applicability, and the design of interchangeable overhangs provides a platform solution for multi-virus detection.