An antibody‐free nucleic acid lateral flow assay ( AF ‐ NALFA ) for visual detection of amplified DNA from Salmonella enterica in milk

Background Food contamination by Salmonella enterica remains a significant public health concern, and its detection in milk still relies on time‐consuming culture‐based methods, whereas faster immunological and molecular approaches are often limited by costs, matrix interference and the need for specialised instrumentation. Aim Here, we developed and evaluated an antibody‐free nucleic acid lateral flow assay (AF‐NALFA), a gold nanoparticle (AuNP)‐based probe‐mediated platform for rapid visual detection of amplified DNA from four S. enterica serovars in pure cultures and artificially contaminated ultra‐high‐temperature (UHT) milk. Methods AF‐NALFA was developed using AuNP‐conjugated oligonucleotide probes for hybridisation‐based detection of S. enterica DNA. The system employed invA ‐specific probes and asymmetric PCR (aPCR) to generate single‐stranded amplicons for direct visual detection. Analytical sensitivity was assessed using synthetic double‐stranded DNA and quantified genomic DNA by qPCR. The assay was further validated in artificially contaminated UHT milk following rapid alkaline lysis, without DNA purification, and compared with plate counting. Major Findings AF‐NALFA enabled specific visual detection within 20 min after amplification. The analytical limit of detection was 10 2 copies/μL for synthetic DNA, consistent with qPCR quantification. In artificially contaminated UHT milk, AF‐NALFA successfully detected S. enterica at the lowest tested bacterial concentration of approximately 1.67 cfu/mL under the evaluated experimental conditions, with signal intensity correlating with DNA copy number and bacterial load. No cross‐reactivity was observed within the bacterial panel evaluated. AF‐NALFA is a sensitive, cost‐effective technique for the visual detection of amplified S. enterica DNA that does not require equipment for the final readout. Scientific and Industrial Implications By combining antibody‐free detection and simplified sample handling, AF‐NALFA provides a promising prototype framework for future development of rapid Salmonella screening assays. More broadly, this platform has potential for future application in industrial milk safety monitoring and quality control.