Journal
APPLIED BIOCHEMISTRY AND BIOTECHNOLOGY
Volume 191, Issue 1, Pages 201-211Publisher
SPRINGER
DOI: 10.1007/s12010-020-03278-x
Keywords
Staphylococcus aureus; Closed-tube loop-mediated isothermal amplification; gyrB gene; pH-sensitive indicator dyes; Visual detection
Funding
- Department of Education, Fujian Province [JA170301]
- Natural Science Foundation of Fujian Province [2018J01452]
- Jimei University National Natural Science Foundation Training Project [ZP2020025]
- Fujian Provincial Department of Science and Technology [2013Y0089]
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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/mu L 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.
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