免疫捕捉real-time PCR对蚜虫中CMV检测体系的建立与应用

Application and Building of Immunocapture Real-time RT-PCR for Detection of Cucumber Mosaic Virus in Individual Peach Aphid (Myzus persicae)

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。

Cucumber mosaic virus （CMV） is transmitted in nonpersistent manner by peach aphid （Myzus persicae）, leading to many plant diseases. The large number of viruliferous peach M. persicae is one of the major culprits for the rampant outbreak of CMV disease in tobacco. In present study, Immunocapture real-time RT-PCR （IC-real-time RT-PCR） was investigated to detect CMV in individual peach aphid （M. persicae） by combining Enzyme-Linked Immunosorbent Assay（ELISA） and conventional RT-PCR. The complex of CMV CP antibodies and CMV particles was adhered to PCR tubes wall, then reverse transcriptase reaction was conducted directly, subsequently real-time PCR. Compared with conventional RT-PCR and ELISA to detect CMV in individual peach aphid, this method was more sensitive and specific. This assay of RT-PCR could be directly carried out without total RNA extraction by capturing CMV particle through CMV CP antibodies, and effectively reduce the pollution and inhibiting effects of proteins, polysaccharide and phenols materials in operating process. Therefore, as a specific, rapid, sensitivity and simple method for detecting slight amount CMV particle, IC-RT-PCR has significance of prediction, forecast and prevention of tobacco virus diseases.