基于锁式探针技术快速检测玉米细菌性枯萎病菌和玉米内州萎蔫病菌  

Rapid Detection of Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis Using a Padlock Probe-Based Assay

作　　者：李志锋[1] 王忠文[1] 冯建军[2,3] 程颖慧[2,3] 李一农[2,3] 章桂明[2,3] 

机构地区：[1]广西大学农学院,广西南宁530004 [2]深圳市检验检疫科学研究院,深圳市外来有害生物检测技术研发重点实验室,广东深圳518045 [3]深圳出入境检验检疫局,动植物检验检疫技术中心,广东深圳518045

出　　处：《生物技术通讯》2016年第4期529-534,共6页Letters in Biotechnology

基　　金：深圳市海外高层次人才创新创业专项资金项目(KQC201109050077A);国家质检总局科研项目(2010IK257);深圳出入境检验检疫科研项目(SZ2011002)

摘　　要：目的:建立一种检测玉米细菌性枯萎病菌和玉米内州萎蔫病菌的方法,为同时检测这2种检疫性细菌提供技术手段。方法:基于靶标序列设计2种检疫性细菌的锁式探针,与靶标菌进行连接消化反应,然后采用通用引物进行滚环扩增,其产物与偶联上对应捕获探针的微球进行杂交,最后通过液相悬浮芯片二重检测。结果:该检测方法能够有效地检测2种检疫性细菌,其检测阈值为103CFU/m L,具有良好的可重复性。结论:建立了一种快速、灵敏的玉米细菌性枯萎病菌和玉米内州萎蔫病菌的二重检测方法。Objective： To establish a method for detecting Pantoea stewartii subsp. stewartii and Clavibacter michiganensis subsp. nebraskensis, provide an effective tool to duplex detection of the two quarantine plant bacterialpathogens on corn. Methods： Based on target unique sequence data of P.stewartii subsp. stewartii and C.michiganensis subsp. nebraskensis, two padlock probes, PSS-PLP and CMN-PLP, were designed, followed by species-spe-cific ligation with target DNA and exonuclease treatment, then amplification of PLPs was carried out, followed byhybridization with COOH beads coupled with relative capture probes, and microarrays were analyzed with Bio-plexliquichip. Results： The two quarantine plant bacterial pathogens could be simultaneously detected using this meth-od we developed, with threshold of 103CFU/ml and good repeatability. Conclusion： We established a rapid, sensi-tive assay to duplex detection of P.stewartii subsp. stewartii and C.michiganensis subsp. nebraskensis on corn.

关 键 词：滚环扩增 玉米细菌性枯萎病菌 玉米内州萎蔫病菌 液相悬浮芯片 

分 类 号：S41[农业科学—植物保护] Q945[生物学—植物学]