Combined systems of recombinase polymerase amplification and membrane immunochromatography or enzyme linked immunoassay for quantitative determination of Salmonella enterica bacterial DNA

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The 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 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 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.

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Sobre autores

T. Serchenya

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Autor responsável pela correspondência
Email: serchenya@iboch.by
Belarus, Minsk, 220084

K. Akhremchuk

Institute of Microbiology of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Belarus, Minsk, 220084

L. Valentovich

Institute of Microbiology of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Belarus, Minsk, 220084

V. Lapina

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Belarus, Minsk, 220084

O. Sviridov

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Email: serchenya@iboch.by
Belarus, Minsk, 220084

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2. Fig. 1. Design diagrams of membrane systems IHA-1 (a) and IHA-2 (b).

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3. Fig. 2. Binding of FAM/Bt amplicons of the invA gene fragment of S. enterica bacteria with an antibody to fluorescein and streptavidin in the IHA-1 (a) and IHA-2 (b) systems: a – 1–10 — DNA amplicon concentration of 0, 0.01, 0.03, 0.05, 0.1, 0.25, 0.5, 1.0, 2.5, and 5 nM; b – 1–8 — DNA amplicon concentration of 0, 0.15, 0.3, 0.5, 1.0, 2.5, 5, and 10 nM.

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4. Fig. 3. Graphs of the concentration dependence of binding of the labeled S. enterica DNA amplicon to streptavidin and antibody to fluorescein in the ELISA-1 (a) and ELISA-2 (b) systems.

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5. Fig. 4. Interaction of S. enterica DNA amplicons (0.015 nM) without additional purification after RPA (I) and purified by column chromatography (II) in the ELISA-1 system: 1 — Typhimurium, 2 — Enteritidis, 3 — London, 4 — Newport, 5 — Derby, 6 — non-pathogenic strain SL7207, 7 — S. enterica DNA standard.

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6. Fig. 5. Binding of FAM/Bt amplicons of the invA gene fragment of different S. enterica serotypes at concentrations of 0.1 nM (I), 0.03 nM (II) and 0.01 nM (III) in the ELISA-1 system: 1 — Typhimurium, 2 — Enteritidis, 3 — London, 4 — Newport, 5 — Derby, 6 — nonpathogenic strain SL7207, 7 — E. coli.

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7. Fig. 6. Determination of the detection limit of S. enterica DNA in combined RPA systems with IHA-1 (a) and ELISA-1 (b): 1–9 — DNA concentration in the RPA reaction mixture 0, 0.5, 5, 50, 500 fg, 5 pg, 50 pg, 500 pg and 5 ng.

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8. Fig. 7. Determination of the detection limit of S. enterica bacteria in milk: a – binding in ELISA-1 of RPA products after the enrichment step for 0–10 h for a milk sample containing S. enterica SL7207 at a concentration of 2 × 100 per 25 g of product; b – binding in ELISA-1 of RPA products from milk samples containing different concentrations of S. enterica SL7207, without the enrichment step: 1–7 – concentration of S. enterica cells in samples per 25 g of milk 2 × 100 (1), 2 × 101 (2), 2 × 102 (3), 2 × 103 (4), 2 × 104 (5), 2 × 105 (6), 2 × 106 (7); K– – negative control sample.

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