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目的:建立一种快速、简单地检测食品中单增李斯特菌的环介导恒温基因扩增方法(loop-mediated isother-mal amplification,LAMP),并初步建立荧光定量LAMP检测单增李斯特菌的方法。方法:根据LAMP方法的原理,设计LAMP检测引物,建立LAMP检测方法,同时对方法的特异性、灵敏度、重复性及初始拷贝数的对数值与反应时间之间的线性关系进行评估。结果:使用LAMP引物可以在0.5h内完成检测工作;LAMP检测技术的灵敏度高出聚合酶链式反应检测技术100倍以上,检出限达到1.72×101拷贝/反应,与其他食源性致病菌无交叉反应,平均实验间变异系数小于5%;反应时间与初始模板浓度的常用对数值有良好的线性关系(R2=0.9994)。结论:本方法具有快速、灵敏、特异、操作简单、设备要求低的特点,具有广阔的应用前景。
OBJECTIVE: To establish a rapid and simple loop-mediated isotherm amplification (LAMP) assay for Listeria monocytogenes in food and to establish a fluorescence quantitative LAMP assay for the detection of Listeria monocytogenes Methods. Methods: According to the principle of LAMP method, LAMP detection primers were designed and LAMP detection method was established. Meanwhile, the linearity between the method specificity, sensitivity, repeatability and initial copy number logarithm and reaction time was evaluated. Results: The detection of LAMP was completed within 0.5h. The sensitivity of LAMP was 100 times higher than that of polymerase chain reaction (PCR), the detection limit was 1.72 × 101 copies / reaction. Compared with other food-borne pathogens No cross-reaction of bacteria, the average coefficient of variation between the experiments was less than 5%; reaction time and initial template concentration commonly used logarithmic values have a good linear relationship (R2 = 0.9994). Conclusion: The method has the characteristics of fast, sensitive, specific, simple operation, low equipment requirements, and has broad application prospects.