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免疫斑点法和免疫捕获RT-PCR检测黄瓜绿斑驳花叶病毒
引用本文:尚海丽,周雪平,吴建祥.免疫斑点法和免疫捕获RT-PCR检测黄瓜绿斑驳花叶病毒[J].浙江大学学报(农业与生命科学版),2010,36(5):485-490.
作者姓名:尚海丽  周雪平  吴建祥
作者单位:浙江大学,农业与生物技术学院,生物技术研究所,浙江,杭州,310029
基金项目:浙江省自然科学基金重点资助项目 
摘    要:黄瓜绿斑驳花叶病毒(Cucumber green mottle mosaic virus,CGMMV)是葫芦科作物上重要的病毒之一.用提纯的黄瓜绿斑驳花叶病毒免疫新西兰大白兔制备CGMMV的多克隆抗体.该多克隆抗体的间接ELISA效价达1∶512 000,与CGMMV17.5 ku外壳蛋白有特异性反应,而与烟草花叶病毒、齿兰环斑病毒和健康植物没有任何反应.用ACP-ELISA分析多抗灵敏度表明多抗检测1∶40 960倍稀释的CGMMV病叶时仍呈阳性反应.用该多抗建立了检测CGMMV的免疫斑点法(dot-ELISA)和免疫捕获RT-PCR方法(IC-RT-PCR).病叶汁液可被dot-ELISA检出的最大稀释度为1∶10 240.IC-RT-PCR从感染CGMMV的病叶组织中扩增到与预期大小相同的480 bp DNA条带,且当CGMMV病叶稀释1∶163 840倍时检测仍呈阳性.检测CGMMV的dot-ELISA和IC-RT-PCR方法的建立为该病毒病的诊断和控制提供了技术支撑.

关 键 词:黄瓜绿斑驳花叶病毒  多克隆抗体  dot-ELISA  IC-RT-PCR

Polyclonal antibody- based dot-ELISA and immunocapture- RT- PCR for Cucumber green mottle mosaic virus detection"
SHANG Hai-li,ZHOU Xue-ping,WU Jian-xiang.Polyclonal antibody- based dot-ELISA and immunocapture- RT- PCR for Cucumber green mottle mosaic virus detection"[J].Journal of Zhejiang University(Agriculture & Life Sciences),2010,36(5):485-490.
Authors:SHANG Hai-li  ZHOU Xue-ping  WU Jian-xiang
Affiliation:Instituteof Biotechnology,College of Agriculture & Biotechnology,Zhejiang University,Hangzhou310029,China
Abstract:Cucumber green mottle mosaic virus (CGMMV) is one of important viruses from cucurbit crops . Using purified CGMMV particles as immunogens,the polyclonal antibody (PAb) was prepared in New Zealand rabbits . The ELISA titer of the PAb was 1∶512000 . The PAb could specifically react with the 17.5 ku coat protein of CGMMV but not with the Tobacco mosaic virus,Odontoglossum ringspot virus and healthy plants . The PAb could detect successfully CGMMV in plant sap at1∶40960 dilution in indirect enzyme-linked immunosorbent assay (ACP-ELISA) . On the basis of the prepared PAb against CGMMV,a dot-ELISA and an immunocapture-RT-PCR (IC-RT-PCR) were established for CGMMV detection . The dot-ELISA could detect CGMMV from infected leaf extract at dilution limit of1 ∶10240 .A specific band with an expected size (480 bp) was amplified from the virus infected leaves by the established IC-RT-PCR,and the IC-RT-PCR could detect the viruses from the virus infected leaf extract at dilution limit of 1∶163 840 . The two detection methods using the prepared PAb provide technical supports for the diagnosis and control of the CGMMV disease .
Keywords:dot-ELISA  IC-RT-PCR
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