两种非放射性标记DNA探针检测菊花矮化类病毒的比较
Comparision Between Two Nonradioactive Labelled DNA Probe for the Detection of Chrysanthemum Stund Viroid
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摘要: 以含菊花矮化类病毒(Chrysanthemum stunt viroid.CSV)全长序列的重组质粒PUC为模板DNA,加入通用引物,在聚合酶链式反应系统中制备地高辛标记的CSV cDNA探针和生物素标记的CSV cDNA探针。用这两种探针检测感染菊花矮化类病毒的菊花样本均为阳性反应,而检测健康对照的结果为阴性。采用PCR-微量板杂交法,地高辛标记的探针检测CSV cDNA的吸光值明显高于生物素标记的探针检测的吸光值,采用斑点杂交法,地高辛标记的cDNAN针能够检测CSV RNA的最低含量为50fg,而生物素标记的cDNA探针能够检测CSV RNA的最低含量为5pg。Abstract: Recombinant PUC inserted with full length nucleic acid sequence of chrysanthemum stunt viroid was used as template DNA to prepare digoxigenin-labelled and biotin-labelled probes with two universal primers by polymerase chain reaction.These two Probes were used to detect Samples infected with CSV.The result showed strong positive signals, while the control of the healthy ones gave negative reaction.Absorbance of digoxigenin-labelled probe was higher than that of biotin-labelled Probe for detecting CSV cDNA with PCR-microplate hybridization.The sensitivity of the digoxigenin-fataled probe for detecting the CSV RNA by dot blot method was 50fg, while that of the biotin-labelled Probe for detectingthe CSV RNA was 5pg.
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Key words:
- CSV /
- rsgoxigenin-labelled probe /
- Biotin-labelled probe /
- Detection
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[3] M.Welnicki and C.Hiruki Highly sensitive digoxigenin-labelled DNA probe for the detection of potato spinder tuber virroid.Journal of Virological Methods.1992,39:91-99 [5] Tatsuji Hataya,Alice Kazuko moue and Eishiro Shikata.A PCR-microplate hybridization method for plant virus detection.journal of Virological Methods,1993,46:223-236 -
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