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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Abstract

The combined bioanalytical systems for the detection ofSalmonella entericabacteria in milk have been developed and studied. These test systems are based on isothermal recombinase polymerase amplification (RPA) of a fragment of theinvAgene 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 ofSalmonella entericasubspeciesenterica, belonging to serogroups B, C, D and E. The detection limit of genomic DNA ofS. entericain the test systems was 0.5 fg. The detection limit ofSalmonella entericabacteria in artificially contaminated milk samples was 8 × 102CFU/ml. After enrichment for 6 h, the detection limit proved to be 2 × 100CFU per 25 g of milk.

About the authors

T. S. Serchenya

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Minsk, 220084 Belarus

K. U. Akhremchuk

Institute of Microbiology of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Minsk, 220084 Belarus

L. N. Valentovich

Institute of Microbiology of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Minsk, 220084 Belarus

V. S. Lapina

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Email: serchenya@tut.by
Minsk, 220084 Belarus

O. V. Sviridov

Institute of Bioorganic Chemistry of National Academy of Sciences of Belarus

Author for correspondence.
Email: serchenya@tut.by
Minsk, 220084 Belarus

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