基于致病基因靶点的PCR法特异性检测土壤青枯菌

Specific detection of Ralstonia solanacearum in soil by PCR-based amplification of pathogenesis-related gene

利用分子生物学手段,以R.solanacearum染色体上的16S rDNA ITS以及毒性质粒携带的致病性相关基因fliC为靶点,分别设计5对序列特异性引物,筛选得到了特异性扩增16SrDNA ITS保守序列的引物对RaITS-1/2以及特异性扩增病菌fliC基因片段的引物对RalfliC-F/R。比较这两对引物的扩增灵敏度、稳定性和特异性可以发现,它们均能够稳定、快速、灵敏地检测青枯菌DNA,检测灵敏度可以达到10 fg DNA/μL。在此基础上成功构建了直接检测土壤青枯菌DNA的PCR检测技术体系。

Ralstonia solanacearum is a typical soil-borne phytopathogen which has extensive hosts,causes severe losses and difficult to control.Sensitive detection of R.solanacearum in soil is essential to disease prediction and management.In this study,5 pairs of specific primers that target R.solanacearum chromosomal 16S rDNA ITS and plasmid-carried pathogenecity related gene fliC were designed and used for molecular detection.2 pairs of primers,RaITS-1/2 and RalfliC-F/R,were screened.RaITS-1/2 can specifically amplify 16S rDNA ITS conserved sequences and RalfliC-F/R can amplify the fliC gene.Results of retrial test on primer sensitivity,stability and specificity revealed that these two primer pairs were sensitive and specific in detection of R.solanacearum DNA in soil.The detection limitation can reach at 10 fg DNA/μL.Thus,the fliC-based PCR detection system for soil R.solanacearum was successfully constructed.This molecular strategy may provide scientific basis for rapidly evaluating soil-borne bacterial wilt risk,designing control plans and resistant variety development.