目的 建立一种准确、快速地检测三七Panax notoginseng根腐病病原真菌茄腐镰刀菌Fusarium solani的实时荧光定量PCR（qRT-PCR）方法。方法 基于茄腐镰刀菌的氨基己二酸还原酶（Lys2）基因设计了qRT-PCR的特异性引物对Fs-QF和Fs-QR，制备了该基因的重组质粒标准品，建立了茄腐镰刀菌的SYBR Green I荧光定量PCR检测方法和实时荧光环介导恒温扩增技术（LAMP）体系。从三七种植区采集到15个具有根腐病、黑斑病典型症状的三七病株及三七种植土壤样品，并提取了这些样品的总DNA，运用建立的检测方法进行检测。结果 建立的qRT-PCR检测方法和实时荧光环介导恒温扩增技术特异性强，能快速检测到携带茄腐镰刀菌的三七根腐病病株。此外，该方法灵敏度高，检测模板质量浓度可低至0.2 pg/μL。结论 建立的方法能明确三七种植土壤以及三七病株中茄腐镰刀菌数量的动态变化。可以为三七土壤处理、根腐病的早期诊断和动态监测以及为带病三七种子、种苗的快速分子检测提供技术支持。

Objective In order to establish an accurate and rapid real-time fluorescence quantitative PCR method for the detection of Fusarium solani, the pathogenic fungus of Panax notoginseng root rot. Methods Based on aminoadipate reductase Lys2 gene of F. solani, specific primers Fs-QF and Fs-QR were designed. The recombinant plasmid standard was prepared and the SYBR Green I fluorescence real-time quantitative PCR method and real-time fluorescent 1oop-mediated isothermal amplication (LAMP) system for detecting F. solani was established. Fifteen samples including P. notoginseng plants with typical symptoms of root rot and black spot as well as soil of P. notoginseng planting area were collected. The total DNA of these samples were extracted as templates, and then detected by the real-time fluorescence quantitative PCR method and 1oop-mediated isothermal amplification established in this study. Results The real-time fluorescent quantitative PCR method and 1oop-mediated isothermal amplification technique established in this study had high specificity. P. notoginseng plants infected with F. solani can be detected quickly. In addition, the method has high sensitivity and the concentration of detection template can be as low as 0.2 pg/μL. Conclusion The method established in this study can be used to reveal the dynamic changes of F. solani concentration in the complex P. notoginseng planting soil and diseased plants. Furthermore, it can provide technical supports for the soil treatment, early diagnosis and dynamic monitoring of root rot, and rapid molecular detection of P. notoginseng seeds and seedlings.

R282.2

国家自然科学基金资助项目（81560610）；云南省科技厅重大专项（2017ZF001）