Locked nucleic acid-enhanced quantitative real-time PCR detection of Ralstonia solanacearum in tobacco planting soil

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.