摘要
目的:针对NDM-1超级耐药菌建立TaqMan-MGB荧光定量PCR快速筛检方法。方法 :根据GenBank登录的NDM-1基因序列设计TaqMan-MGB荧光定量PCR引物与探针。分别优化TaqManMGB荧光定量PCR反应条件和反应体系,并对已知特性的41株标准株、1株NDM-1阳参克隆株以及715株不同地区上送的地方株进行检测,验证方法的特异性、敏感性。结果 :方法特异性好,除阳参克隆株能扩增出阳性对应条带,其它标准株及地方上送株均检测不出相应条带;方法敏感性好,检测限为8 cfu/反应;TaqMan-MGB荧光定量PCR法无需凝胶电泳、紫外观察,实现反应及产物检测一步完成,从菌株核酸提取至检测完成仅需1.5 h-2 h。结论 :本研究针对NDM-1超级耐药菌建立的TaqMan-MGB荧光定量PCR方法,其特异性、灵敏度均相对较好,可应用于针对NDM-1超级耐药菌的监测防控以及应急检测。
Objective： To develop a rapid method based on TaqMan - MGB real time PCR for NDM - 1 （ New Delhi metallo - [3 - laetamase 1 ） superbug screening. Methods： Primers and TaqMan - MGB probe were designed based on the sequences of NDM - 1 from GenBank and the reaction conditions of real time PCR were optimized. For- ty -one standard strains, 1 positive clone strain and 715 strains collected from different regions were tested for the specificity and sensitivity of real time PCR. Results： No strain was found positive except the positive clone strain, and the detection limit of the method was 8 efu/reaction. The test can be finished in 1.5 h - 2 h from DNA extrac- tion to results identification. Conclusion： The specificity and sensitivity of the real time PCR for the detection of su- perbug NDM - 1 gene appeared pretty good. It could be applied to prevention and control of NDM - 1 superbug sur- veillance, as well as emergency detection and identification in the labs.
出处
《中国卫生检验杂志》
北大核心
2013年第8期2000-2003,共4页
Chinese Journal of Health Laboratory Technology
