质粒消除方法及消除效果的评价研究

Study of plasmid curing methods and appraisal of plasmid elimination effect

目的:比较不同质粒消除方法的消除效果。方法:分别以十二烷基磺酸钠、十二烷基硫酸钠、紫外线处理大肠埃希菌,将电泳检测质粒消除菌株PCR扩增检测Ⅰ类整合子,检测Ⅰ类整合子存在情况,并检测未检出Ⅰ类整合子的菌株质粒是否恢复。结果:十二烷基硫酸钠法消除率为27%,十二烷基磺酸钠法消除率7%,紫外线消除率为0。结论:同一菌株的质粒对不同消除剂敏感性不同,同种质粒消除方法对不同的菌株质粒消除效果不同;质粒消除试验后的菌液需接种平板排除未消除菌株的影响;判断质粒是否消除应该用灵敏度高的PCR法检测质粒上的特异性片断或通过检测质粒决定的生物学特性。

Objective：To compare the effect of plasmid elimination when three methods are used to eliminate Escherichia coli plasmids and find which one has the best effect. Methods：Sodium dodecyl sulphate（SDS） and ultraviolet radiation were used respectively to deal with Escherichia coli, and plasmid DNA was extracted and isolated to compare the eliminate effect. The class Ⅰintegron was checked by PCR. The strains were cultured those plasmid DNA without class Ⅰ integron and checked whether the plasmid came back. Results ：The curing frequency of SDS was 27% and 7%. Ultraviolet radiation could not eliminate plasmid effectively. Conclusion ：The curing frequencies are absolutely different while using different curing agents to deal with the same bacterium, and the curing frequencies are also different while using the same curing agent to deal with different bacteriums. The tried strains must be inoculated to plat to filtrate the strains which the plasmids have been thoroughly eliminated. The method checking whether the plasmids are eliminated must be used to check the peculiarly integron of the plasmid or the biologic characteristic determined by the plasmid.