贝氏柯克斯体可视化等温扩增法建立

发布时间：2018-11-03 21:33

【摘要】：目的建立针对贝氏柯克斯体的快速可视化环介导等温扩增(LAMP)方法。方法体外合成贝氏柯克斯体的IS1111a基因,构建重组质粒。利用在线引物设计软件设计3组LAMP引物,用实时浊度仪筛选出最佳引物,同时对羟基萘酚蓝浓度进行优化,建立可视化LAMP反应体系,并评价其敏感性和特异性。结果建立的可视化LAMP反应可准确检测出贝氏柯克斯体的重组质粒和新桥株,不与其他立克次体产生交叉反应,最低可检测到3.6×10~2拷贝/反应,较普通聚合酶链反应(PCR)法的灵敏度提高了一个数量级,等同于实时浊度法和实时荧光定量PCR方法的检测灵敏度。结论建立的可视化LAMP方法可敏感、特异地检测出贝氏柯克斯体感染,操作简单快捷,适用于基层卫生防疫和疫情现场的病原筛查。
[Abstract]:Objective to establish a rapid visualized loop-mediated isothermal amplification (LAMP) method for Coxella bassii. Methods the IS1111a gene of Coxella bassii was synthesized in vitro and the recombinant plasmid was constructed. Three sets of LAMP primers were designed with online primer design software. The best primers were screened by real-time turbidimetry. The concentration of hydroxynaphthol blue was optimized to establish a visual LAMP reaction system, and its sensitivity and specificity were evaluated. Results the established visual LAMP reaction could accurately detect the recombinant plasmid and the new bridge strain of Coxella bassii without cross reaction with other Rickettsiae strains, and the minimum was 3.6 脳 10 ~ 2 copies / reaction. The sensitivity of PCR (PCR) method is increased by one order of magnitude, which is equivalent to that of real-time turbidimetric method and real-time fluorescence quantitative PCR method. Conclusion the established visual LAMP method is sensitive and can be used to detect Coxella basicus infection in different places. It is simple and quick to operate and suitable for pathogen screening in primary health epidemic prevention and epidemic field.
【作者单位】：南京军区军事医学研究所疾病预防控制所;
【基金】：国家重大传染病防治专项(2013ZX10004103;2013ZX10004218) 江苏省科技支撑计划(社会发展)项目(BE2013603) 军队重点项目(BWS14J025)
【分类号】：R440;R535

【相似文献】