A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus.

Jing Xiong, Bei Huang, Ji-Song Xu, Wen-Shu Huang

Applied biochemistry and biotechnology 2020 May

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Food-borne diseases induced by Staphylococcus aureus contamination seriously affect human health and food safety. Therefore, a closed-tube loop-mediated isothermal amplification (LAMP) assay for the visual detection of S. aureus was developed in this study. Firstly, two pairs of outer and inner primers were designed targeting on a conserved fragment of gyrB gene in different S. aureus strains. Secondly, the weakly buffered gyrB-LAMP assays were optimized under various pH values and other conditions, followed by the visual evaluation of five pH-sensitive indicators, and the cresol-red was chosen as the best dye for the best visual performance. Thirdly, the cresol-red-based LAMP assay showed good sensitivity with the detection limit of 5.4 copies/μL for purified DNAs, and good specificity with no cross-reaction with other related species. The specificity of the amplified products was further confirmed by XbaI restriction enzyme digestion analysis. Finally, the cresol-red-based LAMP assay was validated by the clinical-dried fish samples inoculated with known numbers of S. aureus and further validated by 20 blind samples. To our knowledge, this is the first report of a closed-tube LAMP assay based on pH-sensitive indicators for the visual detection of the food-borne S. aureus by the gyrB gene.

Citation

Jing Xiong, Bei Huang, Ji-Song Xu, Wen-Shu Huang. A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus. Applied biochemistry and biotechnology. 2020 May;191(1):201-211