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本研究根据向日葵白锈病菌大亚基核糖体RNA基因序列,向日葵黑茎病菌的ITS-5.8S r RNA基因序列,分别设计特异性DPO(dual priming oligonucleotide)引物,建立同时检测这两种检疫性病菌的多重DPO-PCR检测方法,并对其特异性和灵敏度进行评价。结果表明,所设计的DPO引物特异性强,仅向日葵白锈病菌和向日葵黑茎病菌可分别扩增出307 bp与388 bp的特异性条带,其他参照菌株及阴性对照均无条带;检测体系对混合模板中向日葵白锈病菌和向日葵黑茎病菌的DNA灵敏度均达0.05 ng/μL;且该检测方法对退火温度不敏感,适用范围广。该方法能够准确、快速的检测向日葵白锈病菌和向日葵黑茎病菌,适合于口岸实验室的快速检测。
In this study, based on the sequence of the big subunit ribosomal RNA gene of sunflower white rust and ITS-5.8S rRNA gene sequence of the black-spotted sunflower, the specific DPO (dual priming oligonucleotide) primers were designed respectively to establish the simultaneous detection of the two quarantine Germ multiple DPO-PCR detection method, and its specificity and sensitivity were evaluated. The results showed that DPO primers were designed to be highly specific. Only 307 bp and 388 bp specific bands were amplified from S. typhimurium and H. pombe respectively, and no bands were detected from other reference strains and negative control. The sensitivity of the system to DNA of sunflower white rust and sunflower stems in the mixed template was up to 0.05 ng / μL. The detection method is insensitive to the annealing temperature and has a wide range of applications. The method can accurately and rapidly detect sunflower white rust bacterium and sunflower black stalk bacterium, and is suitable for the rapid test of port laboratory.