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.

中文翻译：

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用于金黄色葡萄球菌视觉检测的封闭管环介导的等温扩增测定。

由金黄色葡萄球菌污染引起的食源性疾病严重影响人类健康和食品安全。因此，在这项研究中，开发了一种用于视觉检测金黄色葡萄球菌的闭管回路介导的等温扩增（LAMP）分析方法。首先，针对不同的金黄色葡萄球菌菌株中的gyrB基因的保守片段设计了两对外部和内部引物。其次，在各种pH值和其他条件下优化了弱缓冲gyrB-LAMP测定，然后对5种pH敏感指示剂进行目测评估，并选择甲酚红作为具有最佳目测性能的最佳染料。第三，基于甲酚红的LAMP分析显示出良好的灵敏度，对纯化的DNA的检出限为5.4拷贝/μL，并且具有良好的特异性，且与其他相关物种没有交叉反应。通过XbaI限制酶消化分析进一步证实了扩增产物的特异性。最后，基于甲酚红的LAMP分析通过用已知数量的金黄色葡萄球菌接种的临床干燥鱼样品进行验证，并进一步通过20个盲样品进行验证。据我们所知，这是基于pH敏感指示剂的闭管LAMP测定法的首次报道，该测定法用于通过gyrB基因目测食源性金黄色葡萄球菌。