嵌套式PCR超灵敏检测马铃薯环腐病菌  

Nested PCR for Ultrasensitive Detection of the Potato Ring Rot Bacterium

作　　者：何云霞[1] 白艳菊[1] 胡林双[1] 于德才[1] 马纪[1] 

机构地区：[1]黑龙江省农业科学院植物脱毒苗木研究所,黑龙江哈尔滨150086

出　　处：《中国马铃薯》2005年第4期204-207,共4页Chinese Potato Journal

基　　金：国家科技部重点基础研究发展规划(973)资助项目(2001CCC02900)

摘　　要：马铃薯种薯中存在环腐病菌潜伏侵染,这种潜伏侵染逐代累积、逐渐表现症状,这是马铃薯环腐病无法根除的根本原因。本研究应用国外成功报道的根据存在于pCS1质粒和环腐菌染色体中一个1.3 kb的插入因子IS1121、高度重复的DNA片段设计的环腐病菌亚种特异性引物序列合成出引物对CMSIF1-CMSIR1和引物对CMSIF2-CMSIR2。以环腐标准菌株、黑龙江省环腐菌株以及马铃薯上其它主要的细菌病原菌(青枯病、软腐病、黑胫病)为试验材料进行直接PCR和嵌套式PCR扩增,结果只有环腐标准菌株和黑龙江省环腐菌株出现特异性片段(直接PCR扩增出1046 bp的片段,嵌套式PCR扩增出864 bp的片段)。将环腐菌纯菌种菌悬液稀释成浓度梯度并与马铃薯组织液混合进行直接PCR和嵌套式PCR检测灵敏度比较,结果表明嵌套式PCR检测灵敏度比直接PCR检测灵敏度提高了100￣1000倍。以明显感病症状的块茎、无明显感病症状的块茎和健康块茎为试验材料进行直接PCR和嵌套式PCR,结果除明显感病症状块茎外,所有无明显感病症状的块茎也均被检测出带有环腐病菌。There is latent infection of Clavibacter michiganensis subsp, sepedonicus in potato tissues. The latent infection accumulates for generations and shows obvious symptoms finally. It is the basic reason that the ring rot of potato can not be eliminated thoroughly. We first prepared the primer pairs CMSIF1- CMSIR1 and CMSIF2-CMSIR2, designed based on the sequence of the 1.3 kb insertion element ISI121 , a high repeated segment of DNA that is present in plasmid pCS1 and in the chromosome of C. m. subsp, sepedonicus, reported successfully. Direct PCR and nested PCR were performed with the standard strains of C. m. subsp, sepedonicus, the strains of C. m. subsp, sepedonicus in Heilongjiang and the other main pathogenic bacteria in potato, and only the strains of C. m. subsp, sepedonicus had specific products. To compare the sensitivities of direct PCR and nested PCR, a serial dilution of the suspension of C. m. subsp, sepedonicus was amplified with the primer pair CMSIF1- CMSIR1 and the direct PCR products were used as templates in nested PCR with the primer pair CMSiF2-CMSIR2. The sensitivity of nested PCR was increased by 100- to 1000-fold. The symptomatic tubers, the symptomless tubers and the healthy tubers were sampled for direct PCR and nested PCR. The results were that all the samples of the symptomless tubers were detected positive.

关 键 词：马铃薯环腐病菌 分子检测 嵌套式PCR 

分 类 号：S435.32[农业科学—农业昆虫与害虫防治]