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建立了实时荧光环介导恒温扩增技术(LAMP)检测罗非鱼中金黄色葡萄球菌(Staphylococcus aureus)的方法,并对方法的灵敏度和特异性进行了评价。针对金黄色葡萄球菌特异性耐热核酸酶ncu基因设计引物进行LAMP扩增。选取LAMP内外引物浓度比、d NTP浓度、Bst聚合酶用量、甜菜碱添加量、扩增温度和扩增时间6个因素进行单因素优化实验,优化确定了其LAMP扩增条件。在优化条件下,该方法的检测限为37 cfu/m L,方法对污染的副溶血弧菌、志贺氏菌、沙门氏菌和大肠杆菌4种病原菌均不呈现非特异性扩增,具有良好的特异性,同时结合实时荧光检测,可以较好地弥补LAMP终点显色无法区分非特异性扩增的缺点。结果表明,该方法用于检测罗非鱼中污染的金黄色葡萄球菌具有灵敏度高、特异性强、操作简便、快速的特点,在食品安全检测方面具有良好的实际应用前景。
A real-time fluorescence ring-mediated isothermal amplification (LAMP) method for the detection of Staphylococcus aureus in tilapia was established. The sensitivity and specificity of the method were also evaluated. Amplified primers were designed according to the ncu gene specific for heat-resistant nuclease of Staphylococcus aureus. Six factors, LAMP concentration ratio, dNTP concentration, Bst polymerase dosage, betaine dosage, amplification temperature and amplification time, were selected to optimize the LAMP amplification conditions. Under the optimal conditions, the detection limit of this method was 37 cfu / m L, and the method showed no specific amplification to the four pathogenic bacteria of pathogenic bacteria such as Vibrio parahaemolyticus, Shigella, Salmonella and Escherichia coli Sex, combined with real-time fluorescence detection, can make up for LAMP endpoint color development can not distinguish the shortcomings of non-specific amplification. The results show that the method is suitable for the detection of Staphylococcus aureus contaminated in tilapia and has the advantages of high sensitivity, specificity, simple and rapid operation, and has good practical application prospects in food safety testing.