黑蜂王台病毒环介导等温扩增检测方法的建立  

Establishment of a real-time loop-mediated isothermal amplification methed for detection of Black queen cell virus

作　　者：王向辉[1] 隋佳辰 张健[1] 郑言[1] 杨倩[1] 宋战昀[2] 王振国[1,2] 

机构地区：[1]吉林农业大学动物科技学院,吉林长春130118 [2]吉林出入境检验检疫局检验检疫技术中心,吉林长春130062

出　　处：《中国预防兽医学报》2015年第9期695-698,共4页Chinese Journal of Preventive Veterinary Medicine

基　　金：国家自然基金项目(31001065);质检总局科技项目(2013IK033)

摘　　要：为建立一种快速检测蜜蜂黑蜂王台病毒(BQCV)的环介导等温扩增方法(LAMP),本研究根据Gen Bank中登录的BQCV-JL1株基因序列(KP119603.1)设计了多套LAMP引物,通过引物筛选,反应体系和反应条件优化,建立了可视化LAMP方法,并且评价了该方法的特异性、敏感性、重复性和稳定性。结果显示,建立的LAMP方法在63℃下可对BQCV核酸进行高效扩增,可视化判定结果与real-time Turbidimeter仪检测结果一致;该方法具有较强的特异性和较高的敏感性(最低检出限量为4.0×102拷贝/μL),为普通PCR的100倍。临床样品检测结果表明LAMP法检出率高于常规PCR。本研究建立的可视化LAMP方法操作简便,适用于BQCV的快速检测。To establish a rapid and sensitive assay for Black queen cell virus （BQCV） detection, a real-time loop-mediated isothermal amplification （LAMP） method was developed with a set of primers targeting the conservative sequence of BQCV VP gene with the LAMP real-time Turbidimeter. Under the optimized reaction conditions including the amplification in a constant temperature at 63℃for 35 min after designed detecting BQCV, The results show that this method can detect the minimum at 4.0×102 copies/μL, even don＇t cross-react with related bee diseases, and with a good repeatability and stability. Clinical trials indicated that LAMP method and general RT-PCR with the same result, but the detection rate of LAMP is much higher than general RT-PCR. Therfore, this establishment of the method provides an effective method for the rapid and specific detection BQCV. Key words： Black queen cell virus; loop-mediated isothermal amplification; detection

关 键 词：黑蜂王台病毒 环介导等温扩增技术 检测 

分 类 号：S852.65[农业科学—基础兽医学]