Nucleic acid chromatography-based colorimetric biosensing platform for rapid and sensitive detection of Salmonella

• An instrument-free assay that couples asymmetric isothermal amplification with immunochromatography achieved 10 cfu per mL Salmonella detection, representing a 100-fold improvement in detection capability; • A double-signal amplification strategy was designed: an asymmetric RPA system first generates numerous ss-amplicons, each of which then offers abundant binding sites for the FAMp and the signal nanomaterial; • The developed approach has good analytical performance, excellent resistance to matrix interference and extensibility; • It provides an alternative tool for field detection pathogen in point-of-care-test settings. The capability to rapidly detect a low load of Salmonella at the early stage of contamination in food samples, especially in field settings, is highly valuable but remains a challenge. Herein, a novel assay was developed by integrating asymmetric isothermal amplification (asyRPA) with immunochromatographic assays (ICA), designated naCA. Sufficient single-stranded amplicons (ss-amplicons) were generated by optimizing the ratio of primers for isothermal amplification, thereby offering abundant binding sites for short-DNA probes and corresponding signal nanoparticles. In this format, the sensitivity was enhanced by over 100-fold compared to the conventional RPA-based assay, with a detection limit as low as 10 cfu/mL. The correlation coefficient (R 2 ) exceeded 0.99 across the range of 10-10 6 cfu/mL in samples spiked with target bacteria. Validation was performed by measuring 17 real samples, with results showing 100% concordance with those obtained by quantitative polymerase chain reaction (qPCR). Importantly, the highly sensitive, user-friendly, and cost-effective naCA platform is not only applicable to large-scale Salmonella screening but exhibits scalability through modification of the conserved fragments of the target gene, offering a powerful tool for field testing in resource-limited scenarios.