甘薯丛枝病植原体的PCR检测  

PCR Detection of Phytoplasma from Sweet Potato Witches’ Broom Disease

作　　者：兰平[1] 李文凤[2] 朱水芳 王振镒[1] 

机构地区：[1]西北农业大学基础科学系,杨凌712100 [2]河北农业大学植保系,保定071001 [3]农业部植检所,北京100029

出　　处：《植物学通报》2001年第2期210-215,共6页Chinese Bulletin of Botany

基　　金：农业部农业生物技术应用研究专项基金

摘　　要：以报道的植原体 (Phytoplasma) 1 6SrDNA基因保守序列为依据 ,设计合成了两对引物对R1 6mF2 /R1 6mR2 和R1 6F2 /R1 6R2 ,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PCR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 .1 0 73ng/μl,在PCR基础上的巢式PCR可以将灵敏度提高约 1 0 0 0 0倍 ,所需模板DNA仅为 0 .0 1 0 73pg/μl,在甘薯丛枝病的检测中是一种快速、灵敏、可靠的方法。Using the software of PCRDESN,two pairs of primers, R 16 mF 2/ R 16 mR 2 and R 16 F 2/ R 16R 2, 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.

关 键 词：甘薯 植原体 取合酶链式反应 巢式PCR 丛枝病原检测 

分 类 号：S432[农业科学—植物病理学]