牛病毒性腹泻病毒LAMP-LFD检测方法的建立及初步应用

Establishment and Preliminary Application of LAMP-LFD Detection Method for Bovine Viral Diarrhea Virus

旨在建立一种用于牛病毒性腹泻病毒(bovine viral diarrhea virus,BVDV)的特异、灵敏、快速、简便的检测方法。试验利用环介导等温扩增技术(loop-mediated isothermal amplification LAMP)与横向流动层析试纸(lateral flow dipstick,LFD)相结合的技术,根据BVDV 5′UTR序列保守区段设计4条LAMP特异性引物和1条异硫氰酸荧光素标记的DNA探针,再利用LFD检测扩增产物。通过对反应温度、反应时间、内外引物浓度比、Bst DNA 2.0聚合酶浓度、dNTP Mix浓度、Mg^(2+)浓度等优化后对该方法的特异性及检测灵敏度进行分析并对52份临床样本进行检测,建立了检测BVDV的LAMP-LFD方法。结果显示:LAMP-LFD可特异性检测BVDV,而口蹄疫病毒、牛流行热病毒、牛疱疹病毒1型等均未检出。LAMP-LFD的敏感性较高,对pUC57-5′UTR质粒最低检测浓度为1.9×10^(1)拷贝·μL^(-1)。LAMP-LFD与PCR两种检测方法均检测出5份阳性,符合率为100%。本试验成功建立了用于BVDV快速检测的LAMP-LFD方法,该检测方法检测时间短,特异性强,操作简单,灵敏性高,可作为一种新型检测方法应用于基层检测。

In order to establish a specific,sensitive,rapid and simple detection method for bovine viral diarrhea virus(BVDV),four loop-mediated isothermal amplification(LAMP)specific primers and one DNA probe labeled with fluorescein isothiocyanate were designed according to the conserved segment of BVDV 5′UTR sequence by combining loop-mediated isothermal amplification(LAMP)with lateral flow dipstick(LFD).By optimizing the reaction temperature,reaction time,ratio of internal and external primer concentrations,Bst DNA 2.0 polymerase concentration,dNTP Mix concentration,Mg^(2+)concentration,etc.,the specificity and detection sensitivity of this method were analyzed,and 52 clinical samples were tested.A LAMP-LFD method for detecting BVDV was established.LAMP-LFD detection technology can specifically detect BVDV,but foot-and-mouth disease virus,bovine epidemic fever virus,and bovine herpesvirus type 1 have not been detected.The sensitivity of LAMP-LFD is high,and the minimum detection concentration is 1.9×10^(1)copies·μL^(-1).Both LAMP-LFD and PCR detection methods detected 5 positive samples,with a coincidence rate of 100%.The LAMP-LFD method for rapid detection of BVDV was successfully established in this experiment.The detection method has the advantages of short detection time,strong specificity,simple operation and high sensitivity,and can be used as a new detection method for grassroots detection.