Recombinase Polymerase Amplification Coupled with Membrane Immunochromatography or Enzyme-Linked Immunoassay for Detection of Pathogenic Bacteria Listeria monocytogenes

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Abstract— Highly sensitive bioanalytical systems have been developed and assessed for the detection of the pathogenic bacteria Listeria monocytogenes, which contaminates food products and causes dangerous foodborne illnesses. These systems integrate recombinase polymerase amplification (RPA) with lateral flow assay (LFA) on test strips or enzyme-linked immunosorbent assay (ELISA) in microplates. The detection limit was 1 fg/µL of L. monocytogenes genomic DNA. The efficiency of DNA extraction was shown to influence the detection limit of the bacteria in the test systems. The bsh gene fragment that was chosen for DNA amplification had not been used for RPA before, and it was found to be specific for all pathogenic L. monocytogenes. Additionally, no cross-reactions with other foodborne pathogens were observed. The minimum assay time was achieved with the use of the test systems. The effectiveness of the test systems for identifying L. monocytogenes in food was validated by using milk samples. The RPA-LFA and RPA-ELISA successfully detected L. monocytogenes cells at concentrations as low as 8 CFU/mL. The detection limit can be reduced to 2 bacterial cells per 25 g of sample after enrichment for 5 h. The proposed test systems are a promising tool for monitoring the potential presence of L. monocytogenes in food products, as they are both sensitive and rapid.

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