A colorimetric microplate assay for determination of Staphylococcus aureus DNA is described. Linear padlock probes were designed to recognize target sequences. After DNA binding, the linear padlock probes were circularized by ligation and then hybridize with biotin-labeled capture probes. Biotin-labeled capture probes act as primers to initiate the RCA. The biotin-labeled RCA products hybridize with digoxin-labeled signal probes fixed on streptavidin-functionalized wells of a 96-well plate. To enhance sensitivity, an AuNP-anti-digoxigenin-POx-HRP conjugate was added to the wells and then bound to digoxin-labeled signalling probes. The oxidation of tetramethylbenzidine (TMB) by H2O2 produces a color change from colorless to blue via HRP catalysis. After the reaction was terminated, absorbance is measured at 450 nm. For target sequences of Staphylococcus aureus, the detection limit is 1.2 pM. For genomic DNA, the detection limit is 7.4 pg.μL−1. The potential application of the method was verified by analyzing spiked food samples. Graphical abstract Schematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity. Schematic representation of rolling circle amplification and functionalized AuNP-based colorimetric determination of Staphylococcus aureus. The method uses streptavidin-functionalized 96-well plates and RCA as a molecular tool and AuNP-anti-digoxigenin-POx-HRP as signal transduction markers to increase sensitivity.

中文翻译：

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基于滚环放大比色法测定金黄色葡萄球菌

描述了一种用于测定金黄色葡萄球菌 DNA 的比色微孔板测定法。线性挂锁探针旨在识别目标序列。DNA 结合后，线性挂锁探针通过连接环化，然后与生物素标记的捕获探针杂交。生物素标记的捕获探针作为引物启动 RCA。生物素标记的 RCA 产品与固定在 96 孔板链霉亲和素功能化孔上的地高辛标记信号探针杂交。为了提高灵敏度，将 AuNP-anti-digoxigenin-POx-HRP 偶联物添加到孔中，然后与地高辛标记的信号探针结合。通过 HRP 催化，四甲基联苯胺 (TMB) 被 H2O2 氧化产生从无色到蓝色的颜色变化。反应终止后，在 450 nm 处测量吸光度。对于金黄色葡萄球菌的靶序列，检测限为1.2 pM。对于基因组 DNA，检测限为 7.4 pg.μL−1。通过分析加标食品样品验证了该方法的潜在应用。图形摘要 金黄色葡萄球菌的滚环放大和功能化 AuNP 比色测定的示意图。该方法使用链霉亲和素功能化的 96 孔板和 RCA 作为分子工具，使用 AuNP-anti-digoxigenin-POx-HRP 作为信号转导标记来提高灵敏度。金黄色葡萄球菌的滚环放大和功能化 AuNP 比色测定的示意图。