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当前位置: 首页 > 文章 > 地高辛标记的cDNA探针检测烟草花叶病毒、黄瓜花叶病毒及马铃薯Y病毒 植物病理学报 2004,34 (1) 75-79
Position: Home > Articles > Digoxigenin-labelled cDNA probes for the detection of Tobacco mosaic virus, Cucumber mo- saic virus and Potato virus Y Acta Phytopathologica Sinica 2004,34 (1) 75-79

地高辛标记的cDNA探针检测烟草花叶病毒、黄瓜花叶病毒及马铃薯Y病毒

作  者：
杜国英;王锡锋;周广和
单  位：
中国农业科学院植物保护研究所
关键词：
TMV;CMV;PVY;地高辛标记探针;斑点杂交
摘  要：
根据已发表的烟草花叶病毒(Tobacco mosaic virus,TMV)、黄瓜花叶病毒(Cucumber mosaic virus,CMV)和马铃薯Y病毒(Potato virus Y,PVY)的外壳蛋白基因序列,设计特异引物,分别以提取的TMV、CMV和PVY侵染的病叶总RNA为模板,反转录PCR进行体外扩增,分别得到长度为0.44、0.77、0.80 kb的目的片段,并克隆到pGEM-T easy质粒载体上,以构建的重组质粒为模板,用PCR方法合成了相应的地高辛标记的双链DNA探针。以合成的探针通过斑点杂交技术检测烟草病叶总RNA和烟草病叶汁液。TMV、CMV和PVY的3种地高辛探针检测各自感染的烟草病叶总RNA的稀释低限分别为1:1 000、1:10 000、1:320,检测各自侵染烟草病汁液的最大稀释倍数分别为1:100、1:100、1:10,而每种探针与健康烟草和其它2种病毒的反应均为阴性。
译  名：
Digoxigenin-labelled cDNA probes for the detection of Tobacco mosaic virus, Cucumber mo- saic virus and Potato virus Y
作  者：
DU Guo-ying , WANG Xi-feng, ZHOU Guang-he (State Key Laboratory for the Biology of Plant Diseases and Pests, Institute of Plant Protection, Chinese Academy of Agricultural Sciences, Beijing 100094, China)
关键词：
TMV; CMV; PVY; digoxigenin-labelled cDNA probe; dot-blot hybridization
摘  要：
The coat protein genes of Tobacco mosaic virus , Cucumber mosaic virus and Potato virus Y were amplified from the extracted RNA of infected tobacco leaves by RT-PCR, using the specific primers designed according to the nucleotide sequences of the coat protein gene of the three viruses. These three fragments were cloned into pGEM-T easy vectors respectively, and then the nucleotide sequences were determined, their lengths were 442,776 and 808 bases respectively. Using these clones, digoxigenin-labelled cDNA probes were synthesized by PCR, and applied in dot-blot hybridization to detect TMV, CMV and PVY. The sensitivity for detection of the total RNA of infected tobacco leaves by TMV, CMV and PVY was 1 : 1 000, 1 : 10 000, 1 : 320 respectively; while that using simple extraction of infected leaves was 1 : 100, 1 : 100 and 1 : 10 respectively. The result showed that the three probes were stable, sensitive and specific.

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