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

The combined bioanalytical systems for the detection of bacteria in milk have been developed and studied. These test systems are based on isothermal recombinase polymerase amplification (RPA) of a fragment of the gene and detection of the DNA amplicons containing biotin and fluorescein residues by a 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, allows to analyze a large number of samples and quantify the result. It has been established that the developed bioanalytical systems are characterized by broad specificity for various serotypes of subspecies , belonging to serogroups B, C, D and E. The detection limit of genomic DNA of in the test systems was 0.5 fg. The detection limit of bacteria in artificially contaminated milk samples was 8 × 10 CFU/ml. After enrichment for 6 h, the detection limit proved to be 2 × 10 CFU per 25 g of milk.