婴幼儿奶粉中克罗诺杆菌DNA快速提取方法的应用

Study on rapid DNA extraction of Cronobacter spp.from infant milk powder

目的探讨婴幼儿奶粉前增菌液中克罗诺杆菌快速、简便、高效、经济的DNA提取方法。方法制备克罗诺杆菌人工污染奶粉前增菌液,比较高速离心和氯仿振荡低速离心去除蛋白和脂质效果及简便性,应用煮沸法(去离子水、TE、TE-SDS)、异硫氰酸胍和硅胶膜吸附试剂盒法提取DNA,以本实验室建立的TaqMan-MGB实时荧光PCR评价效果。结果氯仿振荡3000 rpm离心前处理效果比高速离心法好,TE-SDS煮沸法仅需3步骤、19 min完成DNA提取,比试剂盒提取法更快速,实时荧光PCR效果与试剂盒法基本一致,并优于去离子水和TE煮沸法。结论氯仿振荡3000 rpm离心后TE-SDS煮沸提取DNA,能满足TaqMan-MGB实时荧光PCR对婴幼儿奶粉前增菌液克罗诺杆菌的检测需求,提高食品安全风险监测的灵敏度和速度。

Objective To investigate a rapid,simple,efficient and economic method for DNA extration of Cronobacter spp.from pre-culture medium of infant milk powder.Methods Simulated contaminated milk pre-culture medium was prepared with Cronobacter spp.,then the convenience and removal effect of protein and fat were compared between high-speed centrifugation and low-speed centrifugation with chloroform oscillation.The DNA was extracted by boilling method based on three solvents including deionized water,TE and TE-SDS,guanidine thiocyanate cleavage method and silica gel membrane adsorption kit method,then the three extraction methods were evaluated by TaqMan-MGB probe-based real-time PCR established in the laboratory.Results The pre-treatment effect of chloroform oscillation centrifugation at speed of 3000 rpm was better than high-speed centrifugation.The real-time PCR effect of boilling method with TE-SDS was better than that of the deionized water and TE,but equal to the DNA extraction kit method.It could be finished only by three steps in 19 min,more simple and faster than the other two DNA extraction kit methods.Conclusion The boilling method with TE-SDS after pretreatment of chloroform oscillation centrifugation at speed of 3000 rpm could satisfy the detection demand for Cronobacter spp.from pre-culture medium of infant milk powder by TaqMan-MGB probe-based real-time PCR,improving the sensitivity and speed of food safety monitoring.