Development and optimization of an antibody-free nucleic acid lateral flow assay (AF-NALFA) as part of a molecular toolkit for visual readout of amplified Listeria monocytogenes DNA.

Listeria monocytogenes is a Gram-positive foodborne pathogen responsible for listeriosis, a severe disease with high mortality in immunocompromised individuals. Rapid and accurate detection in food samples is essential for food safety. In this study, we developed and optimized an Antibody-Free Nucleic Acid Lateral Flow Assay (AF-NALFA) as part of a molecular detection toolkit for the visual readout of amplified L. monocytogenes hlyA gene, in combination with ultra-fast asymmetric PCR (aPCR) and oligonucleotide probe hybridization. Three critical parameters were optimized: oligonucleotide probe concentration on test and control lines, gold nanoparticle-probe conjugation ratio, and running buffer composition. In pure bacterial cultures, the limit of detection (LOD) of AF-NALFA was 12.62 copies for L. monocytogenes ATCC 7644, 8.68 copies for ATCC 19117, and 4.83 copies for ATCC 13932. These values were quantitatively assessed using qPCR, confirming the assay's consistency in detecting low DNA copy numbers. The prototype demonstrated 100% specificity against 13 other bacterial species. Furthermore, it was successfully tested in artificially contaminated UHT milk after 1 year of storage at room temperature, detecting L. monocytogenes at 1-30 CFU/mL without DNA purification or selective enrichment. The AF-NALFA enabled visual detection of target ssDNA hybridization within 20 min, offering a rapid, cost-effective alternative to DNA detection methods requiring expensive equipment, specialized expertise, and time-consuming procedures. These findings highlight AF-NALFA's potential as a complementary tool for L. monocytogenes surveillance, providing a practical solution for rapid screening in food safety laboratories and epidemiological monitoring.