马铃薯类病毒(PSTVd)非放射性标记cDNA探针制备及其在检测上的应用  

Preparation and application of non-radioactive, biotin-labelled PSTVd-cDNA

作　　者：吕典秋[1] 李学湛[1] 于德才[1] 胡林双[1] 杨希才[2] 

机构地区：[1]农业部脱毒马铃薯种薯质量监督检验测试中心,哈尔滨150086 [2]中国科学院微生物研究所,北京100080

出　　处：《植物病理学报》2005年第2期104-108,共5页Acta Phytopathologica Sinica

基　　金：国家科技部基础研究重大项目(2001CCC02900)

摘　　要：利用含PSTVd单体克隆的重组质粒pGEM PSTVd,通过PCR扩增技术,用生物素标记制备cDNA探针,进行杂交反应检测PSTVd,其中通过化学颜色反应进行判读灵敏度可达50pg,而用化学发光反应进行判读灵敏度可达5pg,分别是R- PAGE检测灵敏度的26倍和260倍,且2种反应特异性和专化性较强。cDNA核酸斑点杂交反应(NASH)检测PSTVd方法准确、灵敏度高,一次检测样品数量多,且对异地样品检测非常方便,是以往其它检测方法的有效补充。pGEM-PSTVd with monomeric PSTVd cDNA (359 bp) was used as a template and the dUTP was partically replaced by biotin-11-dUTP. The cDNA probe was prepared by PCR amplification technique. Colori-metric and chemiluminescent detection for biotinylated probe were used in this test. They were capable of detecting 50 pg purified total RNA by colorimetric assay and 5 pg by chemiluminescent assay. Nonradioactive probes produced with PCR amplification are particularly suitable for practical diagnosis, as they are sensitive and can be rapidly prepared in large quantities. This two assay methods were 26 times and 260 times as sensitivity as R-PAGE respectively. The diagnosis was sensitive, specific, quick and easy to follow.

关 键 词：马铃薯类病毒 探针 核酸斑点杂交 PCR 

分 类 号：S432.41[农业科学—植物病理学] S435.32[农业科学—农业昆虫与害虫防治]