甘薯丛枝病植原体的PCR检测

以报道的植原体（Phytoplasma)16SrDNA基因保守序列为依据，设计合成了两对引物对R16mF2/R16mR2和R16F2／R16R2，以甘薯丛枝病（SPWB）带病植株的叶脉中提取的DNA为模板，应用聚合酶链式反应（PCR）技术和巢式PCR（Nested-PCR)技术对甘薯丛枝病病原进行分子检测。结果表明PCR扩增出了1．5kb的特异片段，在PCB基础上的巢式PCR扩增出了1．2kb的特异片段，灵敏度实验显示该方法所需PCR模板DNA量为0．1073ng/ul在PCR的基础上的巢度PCR可以将灵敏度提高约10000倍，所需模板DNA仅为0．01073pg/ul，在甘薯丛枝病的检测中是一种快速，灵敏，可靠的方法。

PCR Detection ofPhytoplasma from Sweet Potato Witches

Using the software of PCRDESN,two pairs of primers, R16mF2/ R16mR2 and R16F2/ R16R2, were designed based on the 16S rDNA sequence of Phytoplasma from Michigen Aster yellows(MIAY), Elm yellows(EY),Canadian peach X-disease(CX),Jujube witches’ broom(JWB)and Cherry lethal yellow(CLY) published in references. DNAs as templates were extracted from sweet potato midrib infected with or without Phytoplasma. Phytoplasma in sweet potato witches’ broom was detected using PCR and Nested-PCR. A Phytoplasma-specific 1.5 kb fragment and a 1.2 kb special fragment were amplified with PCR and Nested-PCR, respectively. The minimal amount of DNA extracted from the infected sweet potato for molecular detection using PCR and Nested-PCR were 107.3 pg/μl and 0.01073 pg/μl. It is showed that the methods of PCR and Nested-PCR were very sensitive, rapid and reliable in detecting sweet potato disease associated with Phytoplasma. Furthermore, Nested-PCR based on the PCR was more sensitive than PCR about 10000 times in detecting phytoplasma in sweet potato witches’ broom. The conclusion is that the molecular detection of Phytoplasma in sweet potato witches’ broom is a better method nowadays.