应用RT-PCR检测温州蜜柑萎缩病毒

RT-PCR detection of Satsuma dwarf virus

温州蜜柑萎缩病是以日本为主的少数亚洲国家柑橘上的重要病毒病害,多数柑橘品种隐症带毒,不易发现,对其早期准确快速检测尤为重要。以毒源植株的叶、枝皮为材料,对提取的总RNA和总核酸进行反转录和PCR扩增,通过SDV特异引物的设计与筛选,反应体系与反应程序的建立与优化,扩增得到一个251bp的特异片段,测序结果与日本Iwanami报道的SDV序列同源性为99.6%。RT-PCR检测体系的灵敏度为100ng,同时应用该检测体系可以全年检测到毒源植株嫩叶、嫩皮、老叶、老皮中的SDV。

Satsuma dwarf virus（SDV） is an important citrus virus mainly occurred in Japan and other Asian countries. Generally most hosts carry the virus without symptom. To facilitate efficiently detecting SDV, especially from the symptomless hosts, new diagnostic methods are in demand. In this study, the total RNAs or total nucleic acids extracted from the tissue of citrus infected with SDV were used as template. The primers were specifically designed and selected. The reaction system of reverse transcriptionpolymerase chain reaction （RT-PCR） has been set up and optimized. A target band of 251 bp was obtained, which has 99.6% of sequence homology compared with the sequence reported by Iwanami in Japan. The sensitivity of RT-PCR system for SDV in this study is 100 ng. Tissues of young leaf, matured leaf, young bark, matured bark are available for detection of SDV throughout the year.