鲤疱疹病毒Ⅱ型RAA-LFD检测方法的建立

Development of Recombinase-Aid Amplification Assay Combined with Lateral Flow Dipstick for Detection of the Cyprinid Herpesvirus-2

为了建立一种简便、快捷的现地检测鲤疱疹病毒Ⅱ型(CyHV-2)的方法,本文联合重组酶介导扩增(RAA)和侧向流试纸条(LFD)技术,在优选的RAA下游引物和探针的5’端分别进行生物素(Biotin)和荧光(FAM)标记,通过对RAA反应时间和温度等条件的优化,建立了可用于现地检测CyHV-2的RAA-LFD方法。该检测方法在34℃~40℃恒温下反应10 min即可实现对CyHV-2目的基因片段的有效扩增,与其他常见水生动物疫病病原脱氧核糖核酸(DNA)无交叉反应,RAA扩增产物可直接用LFD肉眼观察,对标准质粒pUC57-CyHV-2的最低检测限为8拷贝/μL,比普通聚合酶链式反应(PCR)灵敏度高出10倍。组内和组间重复性试验表明该方法有良好的重复性和稳定性,使用该方法对45份临床样本进行检测,与普通PCR结果符合率为95%。本文建立的RAA-LFD检测方法特异性强、灵敏度高、重复性好、操作方便,可用于CyHV-2的临床快速检测。

In order to develop a rapid and simple assay to detect the cyprinid herpesvirus-2(CyHV-2)on-site,a recombinase-aid amplification(RAA)combined with lateral flow dipstick(LFD)assay is established to label with biotin and FAM at the preferred RAA downstream primer and the 5’-end of the probe.Through the optimization of RAA reaction time and temperature conditions,Ⅱcarp herpes virus type(RAA-LFD CyHV-2)method is established,which can be used for in situ detection.The RAA-LFD complete the detection in 10 minutes at 34℃to 40℃and the detection results could be visualized by naked eyes.The established CyHV-2-specific RAA-LFD is specific detection of CyHV-2,and have no cross-reactions with other poultry common pathogens.The assay is able to detect 8 copies/μL of CyHV-2 genome DNA,which is higher than PCR.Moreover,we have tested 45 clinical samples by RAA-LFD assay and PCR.Our results shows 95%of clinical samples are consistent with in two assay.Therefore,the developed RAA-LFD is a convenient,specific,sensitive and rapid method for the field diagnosis of CyHV-2 infection.