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由黄瓜花叶病毒(Cucumber mosaic virus,CMV)引起的病毒病是烟草上最主要的病害之一,高虫口密度的带毒蚜虫的迁入是烟草上CMV病害发生的主要致病因子。本研究结合当前CMV的两个检测体系——ELISA和real-time RT-PCR,通过CMV抗血清与带毒蚜虫研磨液中的病毒粒体结合反应,病毒复合体粘附在PCR管壁上,然后直接进行反转录反应,随后进行real-time PCR检测,建立了免疫捕捉real-time RT-PCR(Immunocapture real-time RT-PCR)检测单头蚜虫体内CMV的实时定量检测技术,与普通real-time RT-PCR和ELISA相比,灵敏性和特异性显著提高。该方法无需RNA提取等步骤,可以有效地减少样品操作过程的污染和蛋白、多糖以及酚类物质等杂质的影响,能够对微量CMV病毒粒体进行准确、快速、特异和灵敏的定量检测,对烟草病毒病的预测预报、无毒种苗的生产和病害防治都具有重要的科学意义。
The virus disease caused by Cucumber mosaic virus (CMV) is one of the most important diseases in tobacco. The migration of the highly aphid-infected aphids is the main virulence factor of CMV in tobacco. In this study, we combined the two detection systems of CMV, ELISA and real-time RT-PCR, through the combination of CMV antiserum and virosomes in the aphid-laden grinding fluid. The virus complex adheres to the wall of PCR tube, Then, real-time PCR was carried out to detect the CMV in vivo. The real-time quantitative RT-PCR was used to detect CMV in single-head aphids. Compared with normal real The sensitivity and specificity of -time RT-PCR were significantly higher than those of ELISA. The method does not need RNA extraction and other steps, which can effectively reduce the pollution of the sample operation process and the influence of impurities such as protein, polysaccharide and phenolic substance, and can accurately, rapidly, specifically and sensitively detect the trace CMV virion. Prediction of tobacco virus disease prediction, production of non-toxic seedlings and disease prevention and treatment are of important scientific significance.