牛病毒性腹泻病毒LAMP检测方法的建立与应用

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50 min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。

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

Since the 5'UTR gene (GenBank No.:AY278459.1) had 4 isolated 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.