炭疽杆菌双重可视化LAMP检测方法的建立  

Development of a duplex loop-mediated isothermal amplification assay for detection of bacillus anthracis

作　　者：刘玉[1] 陈文琦[2] 王平[1] 操敏[1] 韩一芳[1] 张琪[1] 张锦海[1] 王长军[1] 

机构地区：[1]南京军区疾病预防控制中心疾控所,江苏南京210002 [2]南京医科大学附属南京第一医院皮肤科,江苏南京210006

出　　处：《东南国防医药》2016年第4期341-345,共5页Military Medical Journal of Southeast China

基　　金：国家科技重大专项(2013ZX10004103004);全军十二五重点项目(AWS11C001;AWS11C009);军队后勤科研重点项目(BWS14J025);国家自然科学基金青年基金(81402619);江苏省科技支撑计划社会发展项目(BE2013603);江苏省自然科学基金青年基金(BK20130083)

摘　　要：目的建立同时检测炭疽杆菌cap A基因、PA基因的双重环介导等温扩增(Loop-mediated isothermal amplification,LAMP)方法,用于防范生物恐怖威胁。方法设计和合成分别针对炭疽杆菌cap A基因、PA基因的引物对,通过优化参数,建立可同时检测cap A基因、PA基因的双重LAMP方法,测试敏感性和特异性,并应用于模拟样本和实战检测。结果双重LAMP的检测cap A基因、PA基因的敏感性均可达到50模板拷贝每反应,并具有良好的特异性,在重大保障活动中得到检验。结论双重LAMP方法具有可以同时筛查、简单快速、灵敏度高等优点,在炭疽杆菌检测方面有良好应用前景。Objective To develop a dupplex loop-mediated isothermal amplification （LAMP） assay for the detection of spe-cific structural genes and virulence genes in bacillus anthracis. Methods Two sets of specific primers were designed and synthesized according to CapA and PA genes of bacillus anthracis. The reaction parameters were optimized to develop the dupplex LAMP assay for the rapid detection of bacillus anthracis. This study also tested the sensitivity and specificity of the LAMP assay, and applied it to test simulated and actual samples. Results A double LAMP assay for rapid detection of bacillus anthracis PA gene （ plasmid pX01） and capA gene （ pX02 plasmid） on visualization methods had been established. The detectable sensitivity of the duplex LAMP was 50 tem-plate copy per reaction, and it had good specificity, stability and reproducibility. Conclusion Considering LAMP’s simplicity in op-eration and high sensitivity, there is potential use in clinical diagnosis and surveillance of bacillus anthracis.

关 键 词：炭疽杆菌 环介导等温扩增 检测 

分 类 号：R378.72[医药卫生—病原生物学]