双重PCR技术检测马铃薯环腐病菌和黑胫病菌方法的建立  

Development of Duplex PCR Assay for Detection of Clavibacter michiganensis subsp.sepedonicus and Pectobacterium atroseptica from Potato

作　　者：韩广涛[1] 杨志辉[1] 朱杰华[1] 赵冬梅[1] 韩彦卿[2] 

机构地区：[1]河北农业大学植物保护学院/河北省农作物病虫害生物防治工程技术研究中心,河北保定071001 [2]中国农业大学农学与生物技术学院,北京100193

出　　处：《中国农业科学》2011年第20期4199-4206,共8页Scientia Agricultura Sinica

基　　金：现代农业产业技术体系建设专项资金(CARS-10-P12)

摘　　要：【目的】利用双重PCR技术快速检测马铃薯环腐病菌(Clavibacter michiganensis subsp.sepedonicus)和黑胫病菌(Pectobacterium atroseptica)。【方法】根据GenBank上发表的马铃薯环腐病菌pCS1质粒上纤维素酶A基因序列,对比近缘种及马铃薯上几种重要病原菌的核苷酸序列,设计并合成了1对特异性引物CMS1/CMS2,将设计的引物与已发表的PCR检测马铃薯黑胫病菌特异性引物ECA1f/ECA2r结合,经过条件优化后,建立了双重PCR体系。【结果】利用引物CMS1/CMS2扩增出了1条913 bp的马铃薯环腐病菌特异性条带。检测灵敏度在DNA水平上达100 fg.μL-1,在细菌数上达105CFU.mL-1。利用双重PCR体系对马铃薯环腐病菌和黑胫病菌进行扩增,可获得913和690 bp的2条特异性条带。检测灵敏度在DNA水平上达600 fg.μL-1,在细菌数上达5×105 CFU.mL-1。【结论】成功建立了双重PCR检测马铃薯环腐病菌和黑胫病菌技术体系,该技术能够同时快速可靠地检测出马铃薯环腐病菌和黑胫病菌。【Objective】The objective of this study is to develop a duplex PCR system to simultaneously and reliably detect Clavibacter michiganensis subsp.sepedonicus and Pectobacterium atroseptica.【Method】 Choosing the cellulose A gene sequence encoded by the native plasmid pCS1 of C.michiganensis subsp.sepedonicus which was published on the GenBank and comparing it with the nucleotide sequence of closely-related species and some pathogens of potato,a specific pair of primers,CMS1/CMS2,was designed and synthesized.A duplex PCR system had been established under the optimized PCR parameters using the combining primers CMS1/CMS2 and ECA1f/ECA2r which was a specific pair of PCR primers to detect P.atroseptica.【Result】 Using CMS1/CMS2 primers,a single unique PCR band of 913 bp was amplified from C.michiganensis subsp.sepedonicus.The detection sensitivity was 100 fg？μL-1 of DNA and 105 CFU？mL-1 of bacteria.Under the duplex PCR system,the 913 and 690 bp PCR bands from P.atroseptica could be specifically amplified.The detection sensitivity was 600 fg？μL-1 of DNA and 5×105 CFU？mL-1 of bacteria.【Conclusion】The duplex PCR system could simultaneously and rapidly detect the two pathogens.

关 键 词：马铃薯 环腐病菌 黑胫病菌 双重PCR 分子检测 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]