Antibody-free nucleic acid lateral flow assays (AF-NALFA) are an established approach for rapid detection of amplified pathogens DNA but can yield inconsistent signals across targets. Since AF-NALFA depends on dual hybridization of probes to single-stranded amplicons (ssDNA), site-specific thermodynamic (Gibbs free energy-ΔG) at probe-binding regions may be crucial for performance. This study investigated how site-specific-ΔG and sequence complementarity at probe-binding regions determine Test-line signal generation, comparing native and synthetic amplicons and assessing the effects of local secondary structures and mismatches. Asymmetric PCR-generated ssDNA amplicons of Listeria monocytogenes, Mycobacterium leprae, and Leishmania amazonensis were analyzed in silico and tested in AF-NALFA prototypes with gold-labeled thiol probes and biotinylated capture probes. T-line signals were photographed, quantified (ImageJ version 1.4k), and statistically correlated with site-specific-ΔG. While native ssDNA from M. leprae and L. amazonensis failed to produce AF-NALFA T-line signals, L. monocytogenes yielded strong detection. Site-specific-ΔG below −10 kcal/mol correlated with reduced hybridization. Synthetic oligos preserved signals despite structural constraints, whereas ~3–4 mismatches, especially at capture probe regions, markedly impaired T-line intensity. The performance of AF-NALFA depends on the synergism between thermodynamic accessibility, site-specific-ΔG-induced site constraints, and sequence complementarity. Because genomic context affects hybridization, target-specific thermodynamic in silico evaluation is necessary for reliable pathogen DNA detection.

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.

Dengue virus (DENV) and Zika virus (ZIKV) are public health threats, causing substantial morbidity and mortality worldwide. The co‐circulation of DENV and ZIKV complicates prevention and control efforts, highlighting the importance of studying cross‐reactive antibodies, vaccines, and immune modulators. This study explores the cross‐reactivity and neutralizing capacity of antibodies against DENV elicited by immunization with a ZIKV protein extract. Female isogenic BALB/c mice were immunized with ZIKV protein extract derived from C6/36 cell cultures. Splenic B lymphocytes from immunized mice were fused with myeloma cells using polyethylene glycol (PEG) to generate hybridomas. The resulting antibodies were assessed using ELISA and plaque reduction neutralization tests (PRNT) against ZIKV and DENV including their structural and nonstructural proteins. Our findings showed that immunization induced a diverse antibody response, including antibodies recognizing epitopes unique to ZIKV as well as epitopes shared with DENV. Notably, neutralizing antibodies targeting conserved epitopes, particularly those shared with DENV‐1, were also identified against other DENV serotypes. These results suggest the presence of cross‐reactive neutralizing antibody responses between ZIKV and DENV, potentially exhibiting serotype‐specific reactivity. Collectively, this study provides experimental evidence of flavivirus cross‐reactivity and contributes to a more refined understanding of humoral responses in settings where these viruses co‐circulate.