烟草青枯病菌巢式PCR检测方法的建立及应用

Development and Application of Nested PCR Assays for Detection of Ralstonia solanacearum in Tobacco

利用细菌16S-23S r DNA内源转录间隔区通用引物L1/L2扩增烟草青枯病菌基因组DNA,并对其扩增产物进行克隆测序,经与近缘种序列多重比对分析后,设计1对特异性引物Rs F/Rs R,用于包括烟草青枯病菌在内的15种不同细菌、5种真菌、3种卵菌基因组DNA的PCR扩增。结果表明:在优化的反应体系与程序条件下,该对引物只能从烟草青枯病菌中扩增出241 bp的特异片段,并通过序列测定验证了其准确性;将引物Rs F/Rs R与细菌通用引物L1/L2进行巢式PCR扩增后,其检测灵敏度在DNA水平上可达0.4 fg/μL,较常规PCR提高1 000倍,表明该对引物能有效地用于烟草组织及土壤中青枯病菌的检测。此结果对烟草青枯病的早期诊断、快速检测及病害流行学研究具有重要意义。

Based on differences in the sequences of 16S-23S ribosomal DNA of Ralstonia solanacearum in tobacco and the other closely related species, a pair of primers RsF/RsR were designed and a PCR assay was developed in the study. Among 47 isolates representing 15 bacteria species including R. solanacearum in tobacco and 8 other fungi and oomycetes species, only a single PCR band of 241 bp amplified with DNA extracted from all isolates of R. solanacearum in tobacco, while other tested isolates had no corresponding band. The detection sensitivity increased 1 000-fold to 0.4 fg/μL genomic DNA by developed a nested PCR procedure with bacterial universal primer pair L1/L2 as the first round primers and RsF/RsR as the second round primers. The results showed that nested-PCR assays provided a rapid and sensitive method for the detection of R. solanacearum from naturally infected tobacco tissues and diseased soil samples. It has great significance for early diagnosis and rapid detection of tobacco bacterial wilt, and the research of disease epidemiology.