DAS-ELISA、RT-PCR和IC-RT-PCR检测葡萄卷叶病毒Ⅲ的比较研究

Comparative Studies on DAS -ELISA,RT -PCR and IC -RT -PCR for Detecting Grapevine leafroll-associated virus-3

在生长季节分3次对部分葡萄品种枝条上部、中部和下部叶片及韧皮部,进行双抗体夹心法(DAS-ELISA)、反转录聚合酶链式反应(RT-PCR)和免疫捕捉反转录聚合酶链式反应(IC-RT-PCR)3种方法检测卷叶病毒Ⅲ(GLRaV-3)的比较。结果表明:DAS-ELISA的检出率明显低于RT-PCR和IC-RT-PCR方法。RT-PCR可以检测出RNA提取液为1:10-4的GLRaV-3病毒,但它受到了提取RNA难以及其它物质(如蛋白质、DNA等)干扰的影响,检测难度增加。从检测的灵敏度来看,RT-PCR和IC-RT-PCR比DAS-ELISA灵敏;并且,IC-RT-PCR比其它两种方法较快,整个过程仅需8h。

The effectiveness of double antibody sandwich enzyme-linked immunosorbent assay(DAS-ELISA),reverse tran-scriptase polymerase chain reaction(RT-PCR)and immunocapture reverse transcriptase polymerase chain reaction(IC-RT-PCR)in detecting grapevine leaf roll associated virus-3(GLRaV-3)in grapevines with infected symptoms and grapevines without infected symptoms was evaluated respectively throughout the growing season.Tissue samples were collected from apical,middle and basal leaves.We found that DAS-ELISA,IC-RT-PCR and RT-PCR detected GLRaV-3in nearly all samples of basal leaves collected throughout the growing season;but the detection of GLRaV-3in the vines without infected symptoms was erratic,regardless of the detection method used,and the results were affected by detecting season and tissue samples.Moreover,we found that DAS-ELISA was less sensitive than RT-PCR and IC-RT-PCR.It was difficult to extract the RNA from the grapevine tissue,and the result was affected by protein,DNA and other molecules of the plant tissue used,which increased the difficulty of detection by RT-PCR.In addition,IC-RT-PCR was faster for detecting GLRaV-3than the other two.