摘要
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。
Oligonucleotide primers Cl(-), C2(+), C3(+),T1(-),T2(+) were designed and synthesized according to the central and left-terminal regions of CEVd-A strain. Using these primers, reverse transcription-polymerase chain reaction (RT-PCR) was developed to detect and analyse citrus excortis viroid in citrus (Citrus L.) and etrog citron (Citrus medico L.) in China. RT-PCR amplified products were analysed by PAGE-silver staining method. Specific DNA bands about 210 bp and 370 bp were observed with primer pair C1/C3 and C1/C2, respectively, from the diseased citrus and etrog citron, while no products was obtained with T1/T2 pair. Under the same conditions, no amplified DNA was detected from nucleic acids of healthy samples with the above three primer pairs. Amplified products were further identified by dot blot hybridization with DIG-la-belled CEVd-cDNA probes. The results indicated that the Chinese isolate of CEVd is different in left-terminal region from the CEVd-A strain. With the established RT-PCR system, full-length CEVd-cDNA could be obtained from approximately 0.1 ng total nucleic acids of infected citrus tissues. This system is suitable for practical diagnosis.
基金
国家"八五"攻关项目资助
