烟草科技2017，Vol.50Issue(12)：14-21,8.
基于锁核苷酸(LNA)增敏的植烟土壤青枯雷尔氏菌定量PCR检测
Locked nucleic acid-enhanced quantitative real-time PCR detection of Ralstonia solanacearum in tobacco planting soil
摘要
Abstract
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.关键词
植烟土壤/青枯雷尔氏菌/锁核苷酸(LNA)/定量PCRKey words
Tobacco planting soil/Ralstonia solanacearum/Locked nucleic acid(LNA)/Quantitative PCR分类
农业科技引用本文复制引用
胡利伟,周汉平,尹启生,宋纪真,牟文君,郭建华,薛超群,冯小虎,奚家勤,张志高,张友武,李琰琰..基于锁核苷酸(LNA)增敏的植烟土壤青枯雷尔氏菌定量PCR检测[J].烟草科技,2017,50(12):14-21,8.基金项目
江西省烟草公司抚州市公司资助项目"烟草青枯病和黑胫病检测方法建立及试剂盒研制"(D2015037). （D2015037）
