环介导等温扩增-无电加热法检测乳中阪崎克罗诺杆菌

Detection of Cronobacter sakazakii in milk by Loop-mediated isothermal amplification without electric heating method

设计了一个无电加热器,利用氧化钙和水的化学反应提供热源,建立一种检测乳中阪崎克罗诺杆菌的环介导等温扩增无电检测体系,同时以阪崎克罗诺杆菌的ITS序列为靶基因,设计特异性引物,使其能够实现快速、高效的现场检测婴儿配方奶粉中阪崎克罗诺杆菌,最终通过琼脂糖凝胶电泳法对检测结果进行判定。利用上述建立的无电检测体系,进行阪崎克罗诺杆菌灵敏度实验并与传统用电加热器法进行对比,在不同环境温度下进行测试。结果表明,利用该无电检测体系和基于传统电加热器检测阪崎克罗诺杆菌纯培养物的灵敏度均为2.6×10~2CFU/m L,且人工污染的婴儿配方奶粉中阪崎克罗诺杆菌的灵敏度均为4.2×10~2CFU/g。该无电检测体系稳定性好,在环境温度为4~37℃时,灵敏度均能达到10~2CFU/g。

A novel detection method had been established to detect Cronobacter sakazakii by Loop-mediated isothermal amplification method coupled with an electric-free heater using calcium oxide and water to provide heat through the LAMP processing.Meanwhile,specific primers were designed based on ITS sequences of Cronobacter sakazakii for the rapid,efficient point-of-care testing on Cronobacter sakazakii of powered infant formula.Finally,the results were determined by agarose gel electrophoresis.Sensitivity of the above method was determined and this non-electric heating detection system was compared with the traditional electric heating method.At the same time,sensitivity experiments were performed at different ambient temperatures.The results showed that the sensitivities of Cronobacter sakazakii in the pure cultures were both 2.6×10 2 CFU/mL by using two types of technologies while those in artificial contaminated milk were both 4.2×10 2 CFU/g.This non-electric heating test system had good stability and the sensitivity of 10 2 CFU/g at environment temperature of 4-37℃.