Direct multiplex recombinase polymerase amplification for rapid detection of S taphylococcus aureus and P seudomonas aeruginosa in food.
Direct multiplex recombinase polymerase amplification for rapid detection of S taphylococcus aureus and P seudomonas aeruginosa in food.
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DOI：
10.22099/mbrc.2021.41503.1664
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发表时间：
2022-03
影响因子：
1.6
通讯作者：
中科院分区：
文献类型：
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食物和饮料中毒对人们的健康是有害的，因为它可以导致发烧、胃痛，甚至死亡。为了快速检测食源性病原体的存在，目前广泛采用传统的PCR检测方法。与此同时，在低温和恒温下进行扩增反应的等温PCR方法最近成为在低资源环境中快速识别病原体的有效和替代手段。金黄色葡萄球菌和铜绿假单胞菌是两种最令人担忧的食源性细菌感染。本文建立了一种基于RPA （Recombinase Polymerase Amplification，重组酶聚合酶扩增）的等温PCR方法，可同时检测金黄色葡萄球菌和铜绿假单胞菌，具有较高的灵敏度和特异性。多重RPA的检出限分别为金黄色葡萄球菌和铜绿假单胞菌基因组DNA的10和30 fg/反应。此外，反应时间缩短至仅25分钟，孵育温度低至39℃。特别是多重RPA反应，可以成功地直接鉴定金黄色葡萄球菌和铜绿假单胞菌细胞，分别低至1和5 CFU/反应，而无需提取DNA基因组。此外，多重RPA可靠地检测了牛奶、果汁和瓶装水样品中的两种食源性细菌。总之，本研究报告的直接多重RPA为检测金黄色葡萄球菌和铜绿假单胞菌的存在提供了一种快速、简单、敏感和有效的替代方法，而不需要昂贵的仪器或训练有素的人员。
Food and beverage poisoning is detrimental to people's health since it can lead to fever, stomachaches, and even death. To rapidly detect the presence of foodborne pathogens, conventional PCR assays are currently widely employed. Meanwhile, isothermal PCR methods, in which the amplification reactions take place at a low and constant temperature, have lately emerged as effective and alternative means for quickly identifying pathogens in low-resource settings. Staphylococcus aureus and Pseudomonas aeruginosa are two of the most concerning foodborne bacterial infections. In this work, an isothermal PCR assay based on the Recombinase Polymerase Amplification (RPA) method was developed to simultaneously detect S. aureus and P. aeruginosa with high sensitivity and specificity. The limit of detection for multiplex RPA was 10 and 30 fg/reaction of S. aureus and P. aeruginosa genomic DNA, respectively. Furthermore, the reaction time was reduced to only 25 minutes, with a low incubation temperature of 39°C. Multiplex RPA reactions, in particular, were successful in directly identifying as low as 1 and 5 CFU/reaction of S. aureus and P. aeruginosa cells, respectively, without the need for DNA genome extraction. Moreover, the multiplex RPA reliably detected the two foodborne bacteria in milk, fruit juice, and bottled water samples. In conclusion, the direct multiplex RPA reported in this work offers a quick, easy, sensitive, and effective alternative approach for detecting the presence of S. aureus and P. aeruginosa without the requirement of a pricey instrument or highly-trained personnel.