马铃薯早疫病菌实时荧光定量PCR检测方法的建立

Development of a real-time fluorescence quantitative PCR assay for detection of Alternaria solani causing early blight of potatoes

马铃薯早疫病主要是由茄链格孢(Alternaria solani)引起的一种常见真菌性病害。本试验依据GenBank中链格孢属的ITS序列(Internal Transcribed Spacer),设计并合成了1对特异性引物ptAsQ-F(5′-TTTGCAATGGCAATCAGCG-3′)/ptAs-R(5′-CCCGCAAG-GGGAGACAAA-3′),以重组质粒为标准品构建实时荧光定量标准曲线。结果表明,建立的实时荧光定量PCR检测方法,引物特异性好,灵敏度高。重复性试验表明,组内和组间变异系数均小于2.51%,显示该方法具有较好的检测稳定性。利用该定量检测技术,可以检测出潜伏侵染于叶片和土壤中的早疫病菌。该方法为马铃薯早疫病田间发病的动态监测以及指导高效防控措施提供了技术支持。

Potato early blight caused by Alternaria solani is a common fungal disease. In this study,a pair of primers ptAsQ-F/ptAs-R were designed according to the ITS sequence of Al- ternaria for detection of Alternaria solani. After optimization of the real-time fluorescent PCR program,a standard curve was established using recombinant plasmid,and meanwhile the sensitivity,specificity and repeatability of the reaction were evaluated. The results showed that Alternaria solani could be differentiated by conventional PCR assay with the developed primer set ptAsQ-F/ptAs-R. Sensitivity of real-time PCR was higher than the conventional PCR. Three-time repeats revealed that the coefficients of variation between the intra-assay and inter-assay were both within 2.51% ,indicating the reproducibility characteristic of the detection method to Alternaria solani. The DNA of the pathogen was detectable in infected leaves and soil by this kind of method. The study provides a rapid, specific and sensitive technique support for early detection and the epidemiological prediction of Alternaria solani.