南方水稻黑条矮缩病毒dot-ELISA检测试剂盒的应用性能验证  

Verification of a dot-ELISA detection kit for southern rice black-streaked dwarf virus

作　　者：王亚琴[1] 姜军 黄德青 周雪平[1] 洪健[1] 吴建祥[1] Wang Yaqin;Jiang Jun;Huang Deqing;Zhou Xueping;Hong Jian;Wu Jianxiang(State Key Laboratory of Rice Biology/Institute of Biotechnology,Zhejiang University,Hangzhou Zhejiang 310058,China;Kaifeng Xiangfu Institute of Agricultural Sciences,Kaifeng Henan 475100,China)

机构地区：[1]浙江大学生物技术研究所/水稻生物学国家重点实验室,浙江杭州310058 [2]开封市祥符区农业科学研究所,河南开封475100

出　　处：《中国植保导刊》2018年第7期34-39,共6页China Plant Protection

基　　金：国家转基因重大项目(2016ZX08009003-001);国家重点研发计划课题(2016YFD0300706);现代农业产业技术体系科研专项(nycytx-001)

摘　　要：以制备的抗南方水稻黑条矮缩病毒(Southern rice black-streaked dwarf virus,SRBSDV)单抗2C2为核心,建立了检测水稻叶片和白背飞虱虫中SRBSDV的dot-ELISA试剂盒。试剂盒的灵敏度分析表明,当SRBSDV感染病叶稀释到1∶10 240倍(w/v,g/m L)、单头携毒白背飞虱稀释到1∶51 200倍(头/μL)时仍能检测到SRBSDV。建立的试剂盒检测感染SRBSDV的水稻和携毒白背飞虱呈阳性反应,而检测感染水稻黑条矮缩病毒、水稻矮缩病毒、水稻条纹病毒、水稻瘤矮病毒、水稻条纹花叶病毒、水稻锯齿矮缩病毒的病叶和健康水稻及非携毒白背飞虱呈阴性反应。试剂盒的田间样品检测结果与RT-PCR方法的检测结果的符合率达到100%,核酸测序和序列比对结果发现RT-PCR检测阳性的样品确实感染SRBSDV。试验结果表明,建立的检测试剂盒能准确、有效地检测田间白背飞虱及水稻样品中的SRBSDV,可为我国南方水稻黑条矮缩病的检测和诊断、预测预警及科学防控提供技术服务。Based on monoclonal antibody（MAb） 2 C2 against southern rice black-streaked dwarf virus（SRBSDV）, a dotELISA detection kit for detecting SRBSDV in rice plants and white-backed planthoppers was developed. Sensitivity analysis indicated that the detection kit could detect minimal viruses in infected rice leaf tissue extracts diluted at 1：10240（w/v, g/m L-1） and in one white-backed planthopper tissue extract diluted at 1： 6400（individual/μL）. The specificity of the kit was confirmed by positive reactions of detection with SRBSDV-infected rice plants and viruliferous white-backed planthoppers,and negative reactions of detection with healthy, rice black-streaked dwarf virus-, rice stripe virus-, rice gall dwarf virus-,rice stripe mosaic virus-, or rice dwarf virus-infected rice plants, or non-viruliferous white-backed planthoppers. Field samples were detected using the developed dot-ELISA kit and RT-PCR respectively, and the detection results indicated that their coincidence rate was 100%. Nucleotide sequencing and sequence blast of the PCR-amplified product proved further that all positive samples detected by dot-ELISA kit were infected with SRBSDV. Those results indicated that the developed kit could accurately and effectively detect SRBSDV in field rice plants and white-backed planthopper samples. Therefore,the established dot-ELISA detection kit for SRBSDV can provide technique supports for the detection and diagnosis,forecasting and early warning and scientific control of southern rice black-streaked dwarf disease.

关 键 词：南方水稻黑条矮缩病毒 单克隆抗体 DOT-ELISA 白背飞虱 

分 类 号：S432.4[农业科学—植物病理学]