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

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Abstract

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.

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Funding

This work was supported by the Department of Education, Fujian Province (JA170301), the Natural Science Foundation of Fujian Province (2018J01452), the Jimei University National Natural Science Foundation Training Project (ZP2020025), and the Fujian Provincial Department of Science and Technology (2013Y0089).

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Authors and Affiliations

  1. Fisheries College, Jimei University, Xiamen, People’s Republic of China

    Jing Xiong, Bei Huang, Ji-song Xu & Wen-shu Huang

  2. Engineering Research Center of the Modern Technology for Eel Industry, Ministry of Education, Xiamen, People’s Republic of China

    Jing Xiong, Bei Huang, Ji-song Xu & Wen-shu Huang

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  1. Jing Xiong
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  2. Bei Huang
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  3. Ji-song Xu
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  4. Wen-shu Huang
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Correspondence to Jing Xiong or Wen-shu Huang.

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The presenting author of this manuscript in ACB2019 is Jing Xiong.

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Xiong, J., Huang, B., Xu, Js. et al. A Closed-Tube Loop-Mediated Isothermal Amplification Assay for the Visual Detection of Staphylococcus aureus. Appl Biochem Biotechnol 191, 201–211 (2020). https://doi.org/10.1007/s12010-020-03278-x

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