牛病毒性腹泻病毒LAMP检测方法的建立与应用  

Establishment and Application of LAMP Detection Method of Bovine Viral Diarrhea Virus

作　　者：李家伟[1,2] 郭利[1] 杨勇[1] 王建科[1] 张淑琴[1] 张加力[2] 程世鹏[1] 

机构地区：[1]中国农业科学院特产研究所,特种动物分子生物学重点实验室,长春130112 [2]吉林农业大学研究生院,长春130118

出　　处：《中国畜牧兽医》2015年第12期3111-3118,共8页China Animal Husbandry & Veterinary Medicine

基　　金：牛呼吸道综合征临床快速诊断试剂盒的研制(20130206024NY);中国农业科学院“创新工程”项目

摘　　要：针对牛病毒性腹泻病毒(BVDV)5′UTR基因(GenBank登录号:AY278459.1)序列设计4条特异性环介导等温扩增(LAMP)引物,特异性识别靶基因序列上4个独立区域,采用LAMP技术,利用实时浊度仪实时检测LAMP反应过程中所产生的焦磷酸镁白色沉淀,实时监测反应液浊度来判断反应结果,实现对扩增反应全过程的监控,建立BVDV的LAMP快速检测方法。通过实时浊度仪在恒温63℃下50min完成检测,对方法的特异性、灵敏度、重复性进行了评价。结果显示,经优化该方法只检测BVDV阳性,特异性强;病毒10-6倍稀释时仍能被检测到,比PCR方法灵敏度至少高100倍;重复性良好。LAMP实时浊度法具有简单、快速、灵敏度高、特异性强的优势,为BVDV的临床检测提供了一种简单快速的试验手段。Since the 5′UTR gene（GenBank No.：AY278459.1）had 4isolated regions,we designed a set of 4 LAMP primers to specifically recognize target gene sequences.This study developed a loop-mediated isothermal amplification（LAMP）method for detecting BVDV,using the pyrophosphate magnesium white precipitate for Real-time detection in LAMP reaction process of turbidity instrument,Real-time monitor liquid turbidity to determine result.The whole reaction lasted only 50 minutes at a constructed temperature of 63℃to evaluate specificity,sensibility and repeatability of the method.The result demonstrated that the LAMP assay could only react with BVDV,its specificity was high;It could detect at least 10-6-fold diluted samples,which was 100 more sensitive than PCR assay;And repeatability was good.The simple,rapid,high siensitivity and specificity LAMP assay was a potential tool for the detection of BVDV in field conditions.

关 键 词：牛病毒性腹泻病毒 环介导等温扩增技术 PCR 

分 类 号：Q78[生物学—分子生物学]