Shanghai Veterinary Research Institute, Chinese Academy of Agricultural Sciences, Shanghai, China.
Department of Electronic Engineering, School of Electronic Information and Electrical Engineering, Shanghai Jiao Tong University, Shanghai, China.
J Appl Microbiol. 2022 Dec;133(6):3741-3754. doi: 10.1111/jam.15811. Epub 2022 Sep 14.
The aim of this study was to develop a novel approach using lateral flow recombinase polymerase amplification (RPA-LF) combined with immunomagnetic separation (IMS) for the rapid detection of Staphylococcus aureus in milk.
Under optimum conditions, the average capture efficiency values for S. aureus strains (10 colony-forming units [CFU] per ml) was above 95.0% in PBST and ~80% in milk within 45 min with 0.7 mg immunomagnetic beads. The RPA-LF assay, which comprised DNA amplification via RPA at 39°C for 10 min and visualization of the amplicons through LF strips for 5 min, detected S. aureus within 15 min. The method only detected S. aureus and did not show cross-reaction with other bacteria, exhibiting a high level of specificity. Sensitivity experiments confirmed a detection limit of RPA-LF assay as low as 600 fg per reaction for the S. aureus genome (corresponding to approximately 36 CFU of S. aureus), which was about 16.7-fold more sensitive than that of the conventional polymerase chain reaction method. When RPA-LF was used in combination with IMS to detect S. aureus inoculated into artificially contaminated milk, it exhibited a detection limit of approximately 40 CFU per reaction.
The newly developed IMS-RPA-LF method enabled detection of S. aureus at levels as low as 40 CFU per reaction in milk samples without culture enrichment for an overall testing time of only 70 min.
The newly developed IMS-lateral flow RPA-LF assay effectively combines sample preparation, amplification and detection into a single platform. Because of its high sensitivity, specificity and speed, the IMS-RPA-LF assay will have important implications for the rapid detection of S. aureus in contaminated food.
本研究旨在开发一种新方法，即使用侧向流动重组酶聚合酶扩增（RPA-LF）结合免疫磁分离（IMS），用于快速检测牛奶中的金黄色葡萄球菌。
在最佳条件下，对于金黄色葡萄球菌菌株（每毫升 10 个菌落形成单位[CFU]），在 PBST 中的平均捕获效率值超过 95.0%，在牛奶中为 45 分钟内约 80%，使用 0.7 毫克免疫磁珠。RPA-LF 检测方法包括在 39°C 下通过 RPA 进行 DNA 扩增 10 分钟，并通过 LF 条带可视化扩增子 5 分钟，在 15 分钟内检测到金黄色葡萄球菌。该方法仅检测到金黄色葡萄球菌，与其他细菌没有交叉反应，具有很高的特异性。灵敏度实验证实，RPA-LF 检测方法的检测限低至每个反应 600 fg 的金黄色葡萄球菌基因组（相当于约 36 CFU 的金黄色葡萄球菌），比传统聚合酶链反应方法灵敏约 16.7 倍。当 RPA-LF 与 IMS 结合用于检测人工污染牛奶中的金黄色葡萄球菌时，其检测限约为每个反应 40 CFU。
新开发的 IMS-RPA-LF 方法可在无需培养富集的情况下，在牛奶样品中检测到低至每个反应 40 CFU 的金黄色葡萄球菌，总检测时间仅为 70 分钟。
新开发的 IMS 侧向流动 RPA-LF 检测方法有效地将样品制备、扩增和检测集成到一个平台中。由于其高灵敏度、特异性和速度，IMS-RPA-LF 检测方法将对污染食品中金黄色葡萄球菌的快速检测具有重要意义。
