减蛋综合征病毒环介导等温扩增检测方法的建立  

Establishment of loop-mediated isothermal amplification method for detecting egg drop syndrome virus

作　　者：董春娜[1,2] 李刚[1] 李伟[1] 张坤[1] 史利军[1] 哈小琴[3] 胡永浩[2] 

机构地区：[1]中国农业科学院北京畜牧兽医研究所,北京100193 [2]甘肃农业大学动物医学院,甘肃兰州730070 [3]兰州军区总医院医学实验中心,甘肃兰州730050

出　　处：《甘肃农业大学学报》2011年第2期1-5,共5页Journal of Gansu Agricultural University

基　　金：国家"863"计划项目(2008AA10Z411);中央级公益性科研院所基本业务费专项(0032007008);北京市科委基金项目(Z07010501780701)

摘　　要：根据GenBank登录的减蛋综合征病毒(EDSV)六邻体基因的保守区,设计了3对环介导等温基因扩增(LAMP)引物,优化反应体系,并对其灵敏性、特异性进行验证.结果表明:LAMP优化后的反应体系为外引物浓度0.1μmol·L-1,内引物浓度0.8μmol·L-1,环引物浓度0.4μmol·L-1,Mg2+浓度8.0 mmol·L-1,dNTP浓度0.8 mmol·L-1,甜菜碱浓度0.5 mmol·L-1,BstDNA聚合酶浓度8 U,最佳反应温度65℃;扩增出的EDSV基因呈特征性梯状条带,显色反应呈绿色荧光;检测EDSV的灵敏度高,是普通PCR检测的10倍,可检测到60个拷贝的基因组DNA;特异性强,与鸡新城疫病毒(NDV)、传染性法氏囊病毒(IBDV)、传染性支气管炎病毒(IBV)、鸡胚致死孤儿病毒(CELO)无交叉反应;该方法重复性好,稳定性高,可准确快速地进行减蛋综合征病毒的检测.In order to develop an efficient,rapid and highly specific loop-mediated isothermal gene amplification（LAMP） assay to detect egg drop syndrome virus（EDSV）,three pairs of primers were designed according to hexon conservative region of EDSV.The LAMP was performed using Bst DNA polymerase large fragment under the effect of isothermal amplification.The LAMP reaction system was optimized and the sensitivity and specificity were tested.The results showed that a batter amplification of LAMP was abtained with the reaction system containing 0.1 μmol·L-1 outer primer,0.8 μmol·L-1 imner primer,0.4 μmol·L-1 ring primer,8 mmol·L-1mg2＋,0.8 mmol·L-1 dNTP,0.5 mmol·L-1 betaine,8 U Bst DNA polymerase,reaction temperature was 65 ℃.The products amplified by LAMP from EDSV were distinctively ladder-like band in agarose gel electrophoresis,and green fluorescence should be seen in colorable reaction.The sensitivity of LAMP assay developed in this study was 10 times higher than that of PCR.It had no cross reaction with NDV,IBDV,IBV and CELO.The LAMP assay was more simple and convenient,which could be used economically and effectively in both laboratorial and pen-side testing.

关 键 词：减蛋综合征病毒 环介导等温扩增 检测 

分 类 号：S852.659.1[农业科学—基础兽医学]