Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples
Recombinase polymerase amplification with polymer flocculation sedimentation for rapid detection of Staphylococcus aureus in food samples
复制标题
重组酶聚合酶扩增结合聚合物絮凝沉降快速检测食品样品中的金黄色葡萄球菌
DOI：
10.1016/j.ijfoodmicro.2020.108691
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发表时间：
2020-10-16
影响因子：
5.4
通讯作者：
Dong, Caiwen
中科院分区：
文献类型：
--
作者：
Hu, Jinqiang;Wang, Yi;Dong, Caiwen
目前，快速、灵敏、方便的金黄色葡萄球菌（S. aureus）目视检测方法十分缺乏。在这项研究中，开发了一种基于重组酶聚合酶扩增（RPA）和聚合物絮凝沉降（PFS）的新型检测方法。采用聚合酶链式反应和RPA方法设计并筛选了针对金黄色葡萄球菌nuc基因的4个正向引物F1、F2、F3、F4和3个反向引物R1、R2、R3的12个有效引物组合。对 RPA 反应条件（包括温度、时间和体积以及 PEG8000 和 NaCl 浓度范围）进行了优化。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污染的食品样品（包括猪肉、牛肉、虾、鱼、奶酪、卷心菜、剩米、鸡蛋、牛奶和橙汁）评估了 RPAPFS 测定的潜在用途。结果表明SA5（F2/R2）组合是最佳候选引物。磁珠与RPA产物吸附的最佳温度范围、最短反应时间和最小体积分别为40~42℃、10min和10μL，最佳PEG8000/NaCl浓度分别为0.2g/mL和2.5M。 RPA-PFS 方法可以检测少至 13 fg 的金黄色葡萄球菌基因组 DNA，并且还对 5 种目标金黄色葡萄球菌以及 27 种非目标食源性细菌具有特异性。 RPA-PFS 对人为污染的食品样品中金黄色葡萄球菌的检测限为 38 CFU/mL (g)。此外，RPA-PFS直接通过肉眼判断，总共耗时不到20分钟。总之，本研究开发的RPA-PFS检测方法是一种快速、灵敏、特异的金黄色葡萄球菌视觉检测方法。
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 RPAPFS 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 degrees C, 10 min and 10 mu L, 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.