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J Integr Plant Biol, 1997, 39 (7): -, Research Article
RT-PCR Amplification and Analysis of Citrus Excortis Viroid
Hu Qin-xue, Lin Mu-lan, Zhang Chun-li, Wu De-xi and Chen Jie
doi:
Abstract
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 tnmscription-polymerase chain reaction (RT-PCR) was developed to detect and analyse citrus excortis vimid in citrus ( Citrus L. ) and etmg citron ( Citrus medica 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.
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