目的 建立一種準(zhǔn)確、快速地檢測三七Panax notoginseng根腐病病原真菌茄腐鐮刀菌Fusarium solani的實(shí)時(shí)熒光定量PCR（qRT-PCR）方法。方法 基于茄腐鐮刀菌的氨基己二酸還原酶（Lys2）基因設(shè)計(jì)了qRT-PCR的特異性引物對(duì)Fs-QF和Fs-QR，制備了該基因的重組質(zhì)粒標(biāo)準(zhǔn)品，建立了茄腐鐮刀菌的SYBR Green I熒光定量PCR檢測方法和實(shí)時(shí)熒光環(huán)介導(dǎo)恒溫?cái)U(kuò)增技術(shù)（LAMP）體系。從三七種植區(qū)采集到15個(gè)具有根腐病、黑斑病典型癥狀的三七病株及三七種植土壤樣品，并提取了這些樣品的總DNA，運(yùn)用建立的檢測方法進(jìn)行檢測。結(jié)果 建立的qRT-PCR檢測方法和實(shí)時(shí)熒光環(huán)介導(dǎo)恒溫?cái)U(kuò)增技術(shù)特異性強(qiáng)，能快速檢測到攜帶茄腐鐮刀菌的三七根腐病病株。此外，該方法靈敏度高，檢測模板質(zhì)量濃度可低至0.2 pg/μL。結(jié)論 建立的方法能明確三七種植土壤以及三七病株中茄腐鐮刀菌數(shù)量的動(dòng)態(tài)變化?？梢詾槿咄寥捞幚?、根腐病的早期診斷和動(dòng)態(tài)監(jiān)測以及為帶病三七種子、種苗的快速分子檢測提供技術(shù)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

國家自然科學(xué)基金資助項(xiàng)目（81560610）；云南省科技廳重大專項(xiàng)（2017ZF001）