Combined Systems of Recombinase Polymerase Amplification and Membrane Immunochromatography or Enzyme-Linked Immunoassay for Quantitative Determination of Salmonella enterica Bacterial DNA

Combined bioanalytical systems for the detection of Salmonella enterica bacteria in milk have been developed and studied. These test systems are based on isothermal recombinase polymerase amplification (RPA) of a fragment of the invA gene and detection of the DNA amplicons containing biotin and fluorescein residues by rapid membrane chromatography on test strips or an enzyme-linked immunosorbent assay (ELISA) in microplates. It was shown that the developed test systems are specific, sensitive, and easy to perform. The RPA procedure requires 20 min at a temperature of 40°C. The immunochromatographic detection of amplicons provides rapid testing within 10 min as well as possible visual recording of the result. ELISA takes 75 min and allows one to analyze a large number of samples and quantify the result. It has been established that the bioanalytical systems developed are characterized by broad specificity for various serotypes of Salmonella enterica subspecies enterica, belonging to serogroups B, C, D, and E. The detection limit of genomic DNA of S. enterica in the test systems was 0.5 fg. The detection limit of Salmonella enterica bacteria in artificially contaminated milk samples was 8 × 102 CFU/mL. After enrichment for 6 h, the detection limit proved to be 2 × 100 CFU per 25 g of milk.