金黄色葡萄球菌重组酶聚合酶扩增方法的建立

Establishment of a recombinase polymerase amplification method for detection of Staphylococcus aureus

为建立基于重组酶聚合酶等温扩增(RPA)技术的金黄色葡萄球菌快速检测方法,本研究根据该菌耐热核酸酶nuc基因的保守序列,设计特异性RPA扩增引物,并经反应条件的优化、特异性及灵敏度检测。结果显示:建立的金黄色葡萄球菌快速检测方法可在37℃孵育35 min特异性检测金黄色葡萄球菌,而对其它病原的检测结果为阴性,特异性较强;对该菌的纯培养物和人工污染该菌的牛奶样品中的最低检出限均为10 cfu,敏感性高。利用该方法和国标培养法同时检测市场购买的牛奶样品和猪肉样品,结果显示二者检测结果一致。本研究建立的RPA方法操作简单、反应快速、特异性强、灵敏度高且不依赖于昂贵的仪器设备,适用于基层实验室检测。

To establish a rapid detection method for Staphylococcus aureus(S. aureus) based on recombinase polymerase amplification(RPA) technology, this study designed specific RPA primers based on the conserved sequence of the thermostable nuclease nuc gene of S. aureus, optimized the reaction conditions, evaluated the specificity and sensitivity. The results showed that the established method specifically identified S. aureus after amplification at 37 ℃ for 35 min, and other pathogens showed negative results, indicating a good specificity. The minimum limit of detection(LOD) of S. aureus culture and artificially contaminated milk samples was both 10 cfu, indicating a high sensitivity. The established RPA method and national standard culture method were employed simultaneously to examine the milk and pork samples bought from market, and the results were consistent with each other.The RPA method established in this study has a rapid reaction process, good specificity and high sensitivity, it is easy to operate, and does not rely on expensive instruments and equipment, and is suitable for detection in grass-roots laboratory.