生物素标记GFV－cDNA探针的制备及在检测葡萄扇叶病毒上的应用  

Detection of Grapevine Fanleaf Virus by Dot-blot Hybridization with Biotin-labelled GFV-cDNA Probe

作　　者：谷洪仓[1] 严敦余[1] 刘焕庭 邱并生[1] 王晋芳[1] 田波[1] 

机构地区：[1]山东农业大学,中国科学院微生物研究所,山东省果树研究所

出　　处：《中国病毒学》1994年第1期48-53,共6页Virologica Sinica

摘　　要：以整合到质粒中的ＧＦＶ－ｃＤＮＡ为模板经ＰＣＲ合成了生物素标记的ＧＦＶ单、双链探针。用合成的探针对提纯的ｃＦＶ－ＲＮＡ＿２、感染ＣＦＶ的昆诺藜叶及１８株而萄进行ＤＮＡ－ＲＮＡ杂交检测表明：检测提纯病毒ＲＮＡ＿２的灵敏度为１．５ｐｇ／斑点，感染ＧＦＶ的昆诺藜提取液最高稀释度可达４０９６０倍；１１株显示典型扇叶症状的样品杂交结果均为阳性，且汁液稀释４００～８００倍仍能测出，７株不显示典型扇叶症状的葡萄中３株受到ＧＦＶ的侵染。单、双链探针最适使用浓度分别为１／２００及１／１００。he 5′and 3'-primers of 21 bp of GFV coat protein gene were synthesized according to the GFV-RNA sequence,The cDNA of GFV ccat protein gene was syntheeized with reverse transcriptase using3′-primer and GFV-RNA extracted from the Durified virus as template,Then the ds-cDNA was amplified by PCR technique employing GFV-RNA_2:ss-cDNA as templates and 5',3′-primers and ligated-with pBluesclpt ks.The ds-cDNA were transformed into Ecoli XLI-Blue cells,g the cDNA extracted from the recombinantclones of E. coli cells.The biotin-labelled ss,ds-probes have been used for the detection of purified GFV-RNA,GFV-infeted C,quivoa and 18 grapevine leaves by nucleic acid dot-blot hybridization,The minimal amount ofpurified GFV-RNA_2 which gave the visible signal was 1.5pg/test dot and the maximum dilution ofGFV-infected C,quinoa extract which gave visible signal was 1:40960;11 samples with typical symptoms were,petive and some of them could give the signal when they were diluted 1:400￣1:800.3samples out of 7 symptomeless leaves were postive,The optimum dilutlon of ss and ds-cDNA probewere 1:100and 1:200.

关 键 词：葡萄扇叶病毒 CDNA探针 生物素 

分 类 号：S432.41[农业科学—植物病理学]