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为了定量检测植烟土壤中青枯雷尔氏菌的数量,有效防控烟草青枯病,构建了一种锁核苷酸(LNA)荧光定量PCR检测方法.以青枯雷尔氏菌细胞色素c基因(Cyt c)(NCBI Genebank序列号:WP_011002214.1)为靶标,采用LNA技术合成覆盖目标片段的引物序列,结合荧光定量PCR技术,进行了植烟土壤中烟草青枯雷尔氏菌数量的快速检测.结果表明,所构建的Cyt c基因标准曲线的R2>0.99,相关性较好.与普通DNA引物相比,采用Cyt c基因LNA引物进行的PCR适用退火温度更高,扩增效率也更高.通过对水稻土发病烟田18个土壤样品的检测,表明青枯雷尔氏菌数量在2.52×104~1.88×107个/g土壤之间.因此,构建的一种基于Cyt c基因的LNA增敏定量PCR检测体系,适用于水稻土等植烟土壤中青枯雷尔氏菌的定量检测,对烟田土壤的提前检测有助于烟草青枯病发生的预警.“,”To detect the amount of Ralstonia solanacearum in tobacco planting soil before bacterial wilt occurrence and effectively control tobacco bacterial wilt, locked nucleic acid-based (LNA-based) quantitative real-time PCR assay was established and used in this study. Taking cytochrome c gene (NCBI Genbank accession number: WP_011002214.1) from Ralstonia solanacearum as the target, its primer sequence covering the target fragment was synthesized with LNA technique. Quantitative real-time PCR was used to quickly detect the amount of Ralstonia solanacearum in tobacco planting soil. The results showed that the standard curve of cytochrome c gene had a good correlation with R2> 0.99. Comparing with conventional DNA primers, LNA primer of cytochrome c gene could increase annealing temperature of PCR reaction and amplification efficiency. The DNA of 18 soil samples from tobacco fields (paddy soil) infected with bacterial wilt were tested and the amount of Ralstonia solanacearum in per gram of soil was 2.52×104-1.88×107. Therefore, LNA-based quantitative real-time PCR assay is suitable for the quantitative determination of Ralstonia solanacearum in paddy soil,and the early detection of tobacco soil will contribute to the forecast of tobacco bacterial wilt.