Rapid and Sensitive Pathogen Detection and Viability Evaluation: A Lateral Flow Assay Based on One-to-Multiple Signal Amplification

Lateral flow assays (LFAs) offer point-of-care pathogen detection but suffer from low sensitivity and an inability to differentiate pathogen viability. To address these challenges, this work integrates a small DNA-binding molecule, asymmetric isothermal amplification, and immuno-LFA techniques in a novel assay, termed NALFIA. Notably, this assay uses a one-to-multiple secondary signal amplification strategy employing single-stranded amplicons (Ss-amplicons) hybridized with multiple short probes (F-probes) and functionalized signal nanospheres (PSred NSs) to enhance the detection sensitivity, achieving an approximately 100-fold improvement in the detection limit over single-probe hybridized LFAs. Moreover, after only 5 min of chromatography, the outcome could be read by the naked eye, markedly boosting the detection efficiency. Validation with 90 real samples demonstrated 100% concordance with quantitative polymerase chain reaction analysis. Crucially, NALFIA is uniquely capable of evaluating the survival status of pathogens, as demonstrated by its application in investigating UV-induced germicidal efficacy through time-dependent viability ratios. In addition to showing good suitability for mass pathogen screening, this highly sensitive and cost-effective platform can be applied to identify live and dead pathogens, thereby enhancing field-deployable detection and antibacterial efficacy assessments.