用双功能磁性纳米标记技术检测产热稳定直接溶血素副溶血性弧菌的实验研究  

Research on the detection of thermostable direct haemolysin Vibrio parahemolyticus with fluorescent magnetic nanoparticles laleling technology

作　　者：向辉[1] 朱海 孙世宏[1] 李颖 王西丽 张汉斌[1] 

机构地区：[1]广州市天河区疾病预防控制中心,广东广州510655 [2]深圳市易瑞生物技术有限公司,广东深圳518102

出　　处：《中国卫生检验杂志》2016年第21期3117-3120,共4页Chinese Journal of Health Laboratory Technology

基　　金：广东省医学科研基金项目(A2013561)

摘　　要：目的通过抗体与磁性荧光纳米颗粒标记的免疫层析技术,建立一种快速检测产热稳定直接溶血素(TDH)副溶血性弧菌的检测方法。方法构建副溶血性弧菌TDH基因片段,将p ET-28a-TDH重组质粒载体转化大肠杆菌表达并制备克隆抗体。抗体与磁性荧光纳米颗粒偶联后,制成免疫层析检测试纸条。将混有不同浓度标准菌株样品与荧光纳米颗粒-单抗偶联物和试纸条共同反应5 min,紫外光下肉眼观察结果。并进行了实验的敏感性、特异性、重复性测试及样品模拟实验。结果转化的重组质粒载体在E.coli BL21(DE3)可稳定高效地表达目的蛋白。并实现了单克隆抗体与磁性荧光颗粒很好的偶联,所得偶联物与阳性菌株反应特异性强、敏感性高,阴性对照菌株无反应。结论实验成功制备了与副溶血性弧菌TDH毒力基因表达产物相结合的磁性荧光纳米颗粒复合物。检测过程操作简单,具有灵敏度高、特异性强、重复性好和检测时间短等特点。Objective By immunochromatography technology and magnetic fluorescent antibody labeled with nanoparticles,to establish a rapid detection method for thermostable direct haemolysin（ TDH）. Methods Construction of Vibrio parahaemolyticus TDH gene fragment was performed,p ET- 28a- TDH recombinant plasmid expression vector was used to transform E. coli and prepare monoclonal antibodies. The antibody and fluorescent magnetic nanoparticles conjugated to prepare immunochromatographic test strip. The sample was mixed with different concentrations of the standard strain and fluorescent nanoparticles- monoclonal antibody conjugates for strip joint reaction of 5 min,with naked eye observation under UV light. And experimental test was conducted for sensitivity,specificity,reproducibility and sample simulation. Results Transformed recombinant plasmid vector in E. coli BL21（ DE3） can be stably express the target protein. The monoclonal antibody and the magnetic fluorescent particles were very good coupling,the coupling and positive strains were highly specific and sensitive,and the negative control strains had no reaction. Conclusion The magnetic fluorescent nanoparticles composites coupling with the TDH virulence gene expression product of V. p was prepared in the experiment. Testing process is simple with high sensitivity,strong specificity,good repeatability and short detection time.

关 键 词：副溶血性弧菌 热稳定直接溶血毒素 荧光纳米颗粒标记技术 免疫层析 

分 类 号：R378[医药卫生—病原生物学]