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参考国外CEVd-A株中央保守区段(C区)和左端区段(T区),设计并合成引物Cl(-)、C2(+)、C3(+)、T1(-)、T2(+)。对感染 CEVd中国分离物的柑桔(Citrus L.)和香橼(Citrus medica L.)总核酸进行了cDNA第一链合成和PCR扩增,其中C1/C3、C1/C2分别能从感病香橼和柑桔总核酸中扩增出210bp和370bp左右的特异DNA,分别相当于CEVd的左半部和全长片段,T1/T2未能扩增出产物;健康香橼和柑桔总核酸中均未能扩增出产物。扩增结果用DIG标记的CEVd-cDNA探针进行了确证。扩增结果说明:CEVd中国分离物在左端T区与CEVd-A株存在差异。PAGE-银染法分析扩增产物表明:建立的RT-PCR方法可从约0.1ng柑桔总核酸中扩增出全长CEVd-cDNA。
The primers (Cl, -), C2 (+), C3 (+), T1 (-) and T2 (+) were designed and synthesized according to the central conservative region (C region) and left region (T region) of foreign CEVd- ). First-strand cDNA synthesis and PCR amplification were performed on total nucleic acids of citrus (Citrus L.) and Citrus medica L. infected with CEVd Chinese isolate, of which C1 / C3 and C1 / C2, Citron and citrus total nucleic acids were amplified 210bp and 370bp specific DNA, respectively, corresponding to the left half and full length fragments of CEVd, T1 / T2 failed to amplify the product; healthy citron and citrus total nucleic acid All failed to amplify the product. The amplification results were confirmed by DIG-labeled CEVd-cDNA probe. The amplification results show that the CEVd isolate in China is different from the CEVd-A strain in the left T region. Analysis of the amplified product by PAGE-silver staining indicated that the established RT-PCR method can amplify full-length CEVd-cDNA from about 0.1 ng of total citrus nucleic acid.