实时荧光定量PCR法检测十字花科细菌性黑斑病菌

为有效防控我国的检疫性有害生物十字花科细菌性黑斑病菌Pseudomonas syringae pv.maculicola在国内的传播与蔓延,通过设计1对特异性引物3539,利用132株靶标和非靶标菌为模板进行PCR扩增,建立了实时荧光定量PCR法,并进行了模拟种子带菌试验。结果显示,引物3539为只针对十字花科细菌性黑斑病菌扩增出的特异性产物;在模拟种子带菌检测中,常规PCR对菌悬液的检测限为10~5CFU/m L,实时荧光定量PCR的检测限为10~3CFU/m L,其中10~8CFU/m L菌液的Ct值最低,为22.90,10~3CFU/m L菌液的Ct值最高,为35.73,且不同浓度菌液间的Ct值均有显著差异;不同带菌率模拟种子的检测结果表明,常规PCR和实时荧光定量PCR能检测到的带菌率分别为0.5%和0.1%。研究表明,实时荧光定量PCR法不仅可用于病种的检测,也可用于病害的早期诊断。

Development of a real-time quantitative PCR assay for the specific detection of Pseudomonas syringae pv. maculicola in crucifers

To effectively control the bacterial pathogen Pseudomonas syringae pv. maculicola (Psm) in crucifers, a real-time quantitative PCR assay for specific detection of Psm was developed. A pair of specific primers 3539 were designed, and 132 target and non-target bacterial strains were used for amplification and validation. The primer 3539 was only specific to the target bacterial strains without false positive or false negative reaction. In the stimulation of the seed detection of Psm, the minimum detection concentrations of seed suspension were 10⁵ CFU/mL and 10³ CFU/mL by conventional PCR and real-time quantitative PCR, respectively. The Ct value of real-time quantitative PCR significantly varied among different concentrations of the suspension, and the Ct value of 10⁸ CFU/mL suspension was the lowest with 22.90, and the Ct value of 10³ CFU/mL suspension was the highest with 35.73. The results of detection for different simulated infection rate of crucifer seeds showed that the conventional PCR and real-time PCR could detect 0.5% and 0.1% infected seeds, respectively. The real-time quantitative PCR not only can be used for the seed detection of Psm but also can be considered for early disease diagnosis of crucifer bacterial leaf spot.