种子种苗上黄瓜绿斑驳花叶病毒IC-RT-PCR检测方法的建立及应用  

Establishment and application of IC-RT-PCR for cucumber green mottle mosaic virus in seeds and seedlings

作　　者：任春梅[1] 杨柳[1] 缪倩[1] 陆芳[1] 季英华[1] 程兆榜[1] REN Chunmei;YANG Liu;MIAO Qian;LU Fang;JI Yinghua;CHENG Zhaobang(Institute of Plant Protection,Jiangsu Academy of Agricultural Sciences,Nanjing 210014,China)

机构地区：[1]江苏省农业科学院植物保护研究所,南京210014

出　　处：《植物保护》2022年第5期212-219,共8页Plant Protection

基　　金：江苏省农业科技自主创新[CX(19)3108];国家公益性行业(农业)科研专项(201303028)。

摘　　要：黄瓜绿斑驳花叶病毒(CGMMV)是一种检疫性植物病毒,种传和农事操作是其主要传播途径,因此种子和种苗的早期检测尤为重要。鉴于种子检测的特殊性及幼苗病毒含量低的特点,本试验通过制备CGMMV单克隆抗体,结合特异性引物,建立了黄瓜绿斑驳花叶病毒的IC-RT-PCR早期检测方法,比较了IC-RT-PCR与DAS-ELISA和RT-PCR方法的特异性、灵敏度,并对实际检测效果进行了评价。在操作程序上,IC-RT-PCR法与DAS-ELISA法一样简便,但灵敏度远大于DAS-ELISA。IC-RT-PCR与RT-PCR均可检出20~25 ng种子上的病毒,且特异性相当,而DAS-ELISA法只可检出10μg种子上的病毒。综上所述,IC-RT-PCR法可简便有效地应用于种子和种苗上CGMMV的检测,为病毒防治的早期干预提供技术支撑。Cucumber green mottle mosaic virus(CGMMV) is a quarantine plant virus,which mainly transmitted by seeds and agricultural operations.Therefore,early detection of seeds and seedlings is particularly important.In view of the particularity of seed and the characteristics of low virus content in seedlings,we established an early detection method,IC-RT-PCR,for CGMMV detection by preparing its monoclonal antibodies and specific primers.The specificity and sensitivity of IC-RT-PCR,DAS-ELISA and RT-PCR were compared,and the actual detection effect was evaluated.IC-RT-PCR was as simple as DAS-ELISA in operation,but its sensitivity was much higher than that of DAS-ELISA.Both IC-RT-PCR and RT-PCR could detect viruses on 20-25 ng seeds with equivalent specificity,while DAS-ELISA could only detect viruses on 10 μg seeds.In conclusion,IC-RT-PCR can be easily and effectively applied to CGMMV detection on seeds and seedlings,which provided technical support for early intervention in virus control.

关 键 词：黄瓜绿斑驳花叶病毒 免疫捕获RT-PCR 灵敏度 特异性 

分 类 号：S41-32[农业科学—植物保护]