PMA结合实时荧光PCR进行玉米细菌性枯萎病菌细胞活性检测初步研究  

Preliminary study on differentiation of live and dead cell of Pantoea stewartii subsp. stewartii using propidium monoazide combined with real-time fluorescent PCR

作　　者：冯建军[1,2] 刘杰[1] 王飞[1] 李秋枫[1,2] 刘新娇[1,2] 李一农[1,2] 章桂明[1,2] 

机构地区：[1]深圳出入境检验检疫局,广东深圳518045 [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

出　　处：《植物检疫》2014年第2期27-32,共6页Plant Quarantine

基　　金：深圳市海外高层次人才创新创业专项资金项目(KQC201109050077A);深圳出入境检验检疫局科技项目(SZ2011002);深圳市科技研发资金项目(CXB201105100088A)

摘　　要：本文将DNA染料PMA选择渗透性和实时荧光PCR特异性相结合,建立了一种快速灵敏检测玉米细菌性枯萎病菌细胞活性的新方法。结果表明,当菌悬液浓度不高于104 CFU/mL时,终浓度为1μg/mL或更高PMA渗透处理后,可以完全抑制死菌DNA的扩增,而活菌DNA的实时荧光PCR检测仍有荧光信号;当菌悬液浓度为105 CFU/mL时,20μg/mL的PMA也可以完全抑制死细胞DNA扩增。该方法能有效区分玉米细菌性枯萎病菌死活细胞,并有助于监测玉米种子中该病菌的活性。Although DNA-based method is a quick and sensitive assay for detecting plant pathogenic bacteria, it cannot be used to differentiate live and dead cells, due to the persistence of DNA from dead cells in the sample, also the dead cells do not cause disease, thus this poses a problem in seed health testing. We describe a combined the DNA- intercalating dye propidium monoazide （PMA）, real-time fluorescent PCR assay for differentiation of dead and live cell of Pantoea stewartii subsp, stewartii（Pss）. Using cell suspension of Pss was treated with PMA before incubated at room temperature and at 100 ℃ for 5 min to kill Pss. After 10 min in the dark, the mixtures were put on ice under 500W halogen lamp for 10 min to allow PMA to be effective, then recorded by the Ct value of real-time fluorescent PCR. The results show that heat-treated suspensions of Pss containing no more than 10^4 CFU/mL was totally inhibited PCR amplification of DNA from dead cells when pretreated with PMA at 1μg/mL or greater, however, there was no effect on the same concentration of live cell suspension. When heat-treated suspensions containing dead cells of Pss at 10^5 CFU/mL was treated with PMA at 20 μg/mL, the DNA from dead cells can also be totally inhibited amplification. This combined PMA, real-time PCR protocol should prove a promising assay for selective quantification of viable cells of Pss and facilitates monitoring the pathogen in maize seeds.

关 键 词：叠氮碘化丙锭 玉米细菌性枯萎病 TAQMAN探针 活性 

分 类 号：S41-30[农业科学—植物保护]