基于数字化环介导等温扩增技术的牛乳中大肠杆菌快速精准定量分析

Rapid detection of Escherichia coli in milk by digital loop-mediatedisothermal amplification

建立了一种数字化环介导等温扩增技术(loop-mediated isothermal amplification,LAMP)用于牛乳中大肠杆菌的精准快速定量。通过向反应体系中加入聚乙二醇单体进行水凝胶交联,随后在65℃进行等温扩增。由于水凝胶基质的限制作用,一个细菌的特定基因序列只会产生一个扩增子荧光亮点,用于计数。因此,可以通过荧光显微镜进行成像并通过扩增子点数目来确定样品细菌浓度。通过对扩增时间、引物浓度、牛血清白蛋白浓度等条件进行优化,得到最优结果。结果表明,该方法具有快速、简便、高效和高灵敏性的特点,检测灵敏度可降至单个细菌,有望成为食品快速检测大肠杆菌的方法。

This study aimed to establish a digital loop-mediated isothermal amplification(LAMP)method to detect Escherichia coli in milk.This experiment was performed by including two polyethylene glycol(PEG)hydrogel monomers into the LAMP assay for hydrogel cross-linking,followed by isothermal incubation at 65℃.Due to the restriction effect of the PEG hydrogel matrix,one bacterium could only produce one fluorescent amplicon dot for simple digital counting.Therefore,the sample bacterial concentrations can be determined based on the number of fluorescent amplicon dots using a fluorescence microscope for imaging.Optimal results were obtained by systematically optimized the experimental conditions,such as amplification time,primer concentration and bovine serum albumin concentration.The result showed that the detection could be completed with sensitivity down to single bacterium.Considering its rapidity,simplicity,efficiency and sensitivity,the developed digital LAMP has an enormous potential for rapid detection of E.coli in food samples.