Combined systems of polymerase chain reaction and a time-resolved fluorescence immunoassay or membrane immunochromatography for quantitative determination of Salmonella enterica bacterial DNA

Four model bioanalytical systems specific for Salmonella enterica have been developed and studied, in which a polymerase chain reaction (PCR) produced a DNA amplicon containing biotin and fluorescein residues. This enabled to immobilize the amplicon on a functionalized solid phase and to label it biospecifically with europium chelate in microplates or gold nanoparticles on a chromatographic membrane. Quantitative detection of the modified DNA was carried out in immunoassay systems by measuring the Eu³⁺ time-resolved fluorescence (dissociation-enhanced lanthanide fluorescence immunoassay, DELFIA) or by photometry of the colored zone on the chromatographic strip (LFA). Three pairs of primers were developed and examined to obtain selected fragments of the invA gene, which is present in the genomes of all pathogenic Salmonella enterica. The fragments proved to be suitable for the test systems. In the microplate DELFIA system, the concentration range of DNA amplicon quantification was found to be 0.01–10.0 nM, and a detection limit was 2 pM. The limit of DNA visual detection in LFA was 0.05 nM. The possibility of testing the amplicons without additional isolation of pure DNA from the reaction mixture was demonstrated. The high specificity of the developed bioanalytical systems for the detection of various Salmonella enterica serotypes was demonstrated.

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