Currently, rapid, sensitive, and convenient visual detection methods for Staphylococcus aureus (S. aureus) are scarce. In this study, a novel detection method based on recombinase polymerase amplification (RPA) and polymer flocculation sedimentation (PFS) was developed. Twelve effective primer combinations derived from four forward primers F1, F2, F3, F4, and three reverse primers R1, R2, R3 targeting the nuc gene of S. aureus were designed and screened by a polymerase chain reaction and RPA methods. RPA reaction conditions, including temperature, time, and volume as well as PEG8000 and NaCl concentrations range, were optimized. Moreover, the specificity and sensitivity of the RPA-PFS assay were further analyzed. Finally, the potential use of the RPA-PFS assay was evaluated using artificially S. aureus contaminated food samples, including pork, beef, shrimp, fish, cheese, cabbage, leftover rice, egg, milk, and orange juice. Results showed that the SA5 (F2/R2) combination was the optimal primer candidate. The optimal temperature range, the shortest time and the minimal volume of RPA reaction were 40–42 °C, 10 min and 10 μL, respectively and the optimal PEG8000/NaCl concentrations were 0.2 g/mL and 2.5 M, respectively, for the adsorption between magnetic beads and RPA products. The RPA-PFS method could detect as little as 13 fg genomic DNA of S. aureus and was also specific for five target S. aureus as well as twenty-seven non-target foodborne bacteria. The limit of detection of RPA-PFS for S. aureus in artificially contaminated food samples was 38 CFU/mL (g). Besides, RPA-PFS has directly been judged by the naked eye and has totally taken less than 20 min. In short, the assay RPA-PFS developed in this study is a rapid, sensitive, and specific visual detection method for S. aureus.

当前，缺乏用于金黄色葡萄球菌（S.aureus）的快速，灵敏和方便的视觉检测方法。在这项研究中，开发了一种基于重组酶聚合酶扩增（RPA）和聚合物絮凝沉淀（PFS）的新型检测方法。来自四个正向引物F1，F2，F3，F4和三个针对金黄色葡萄球菌nuc基因的反向引物R1，R2，R3的十二种有效引物组合通过聚合酶链反应和RPA方法设计和筛选。优化了RPA反应条件，包括温度，时间和体积以及PEG8000和NaCl浓度范围。此外，进一步分析了RPA-PFS测定的特异性和敏感性。最后，使用人工金黄色葡萄球菌评估了RPA-PFS分析的潜在用途被污染的食物样本，包括猪肉，牛肉，虾，鱼，奶酪，白菜，剩饭，鸡蛋，牛奶和橙汁。结果表明，SA5（F2 / R2）组合是最佳的引物候选。RPA反应的最佳温度范围，最短时间和最小体积分别为40-42°C，10分钟和10μL，最佳PEG8000 / NaCl的最佳吸附浓度分别为0.2 g / mL和2.5M。在磁珠和RPA产品之间。RPA-PFS方法可检测到仅13 fg的金黄色葡萄球菌基因组DNA，并且对5个目标金黄色葡萄球菌以及27个非目标食源性细菌具有特异性。金黄色葡萄球菌RPA-PFS的检出限人工污染的食品样品中的总胆固醇为38 CFU / mL（g）。此外，RPA-PFS可以直接用肉眼判断，整个过程不到20分钟。简而言之，这项研究中开发的RPA-PFS测定法是一种快速，灵敏，特异的金黄色葡萄球菌视觉检测方法。