利用TaqMan探针实时荧光PCR方法检测香石竹细菌性萎蔫病菌

Detection of Burkholderia caryophylli by TaqMan real-time fluorescent PCR

根据香石竹细菌性萎蔫病菌基因组16S-23S rRNA保守序列,设计并合成了一对特异性引物和一条具有稳定点突变特异性探针,建立了对香石竹细菌性萎蔫病菌的TaqMan实时荧光PCR检测方法。除香石竹细菌性萎蔫病菌外,还对其他7种病原细菌菌株进行了荧光PCR检测。结果表明,只有香石竹细菌性萎蔫病菌产生荧光,其他病原细菌均没有荧光产生。与常规PCR相比,实时荧光PCR检测特异性强,灵敏度高,能检测到浓度为0.4 pg/μL的DNA,且能直接用于苗木等样品的检测,适合病害的快速诊断和口岸检验检疫应用。

TaqMan real-time PCR was developed to detect Burkholderia caryophylli. A pair of primers and a probe specific for B. caryophylli were designed according to its intergenic spacer region of 16S-23S ribosomal RNA. Among seven tested pathogenic bacteria, only B. caryophylli was detected. The results showed that a limit of 0.4 pg/μL of B. caryophylli DNA was detected, more specific and sensitive than routine PCR. The method can also apply to the detection of B. caryophylli in inoculated samples and it is useful for rapid diagnosis and quarantine purposes.