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浙江大学学报（农业与生命科学版）
浙江大学学报(农业与生命科学版)  2010, Vol. 36 Issue (5): 485-490    DOI: 10.3785/j.issn.1008-9209.2010.05.002
生物科学与技术     
免疫斑点法和免疫捕获RT-PCR检测黄瓜绿斑驳花叶病毒
尚海丽,周雪平,吴建祥;
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;
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
Instituteof Biotechnology,College of Agriculture & Biotechnology,Zhejiang University,Hangzhou310029,China
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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1:512000,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1:40960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1:10240。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1:163840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 。
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 .
出版日期: 2010-09-13
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尚海丽
周雪平
吴建祥

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.

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". Journal of Zhejiang University: Agric. & Life Sci., 2010, 36(5): 485-490.

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http://www.zjujournals.com/agr/CN/10.3785/j.issn.1008-9209.2010.05.002    或    http://www.zjujournals.com/agr/CN/Y2010/V36/I5/485

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