三七根腐病菌尖孢镰刀菌的Real-time PCR检测方法

doi:10.7668/hbnxb.20190370

摘要/Abstract

摘要： 为了建立一种准确、快速检测三七根腐病病原真菌尖孢镰刀菌的实时荧光定量PCR方法，基于尖孢镰刀菌脂肪酸ω-羟化酶基因设计了实时荧光定量PCR的特异性引物对Fo-QF和Fo-QR，制备了该基因的重组质粒标准品，建立了尖孢镰刀菌的SYBR Green Ⅰ荧光定量PCR检测方法。从三七种植区采集到14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，运用本研究建立的荧光定量PCR方法进行了检测。结果显示，本研究建立的实时荧光定量PCR检测方法特异性强，脂肪酸ω-羟化酶基因只以尖孢镰刀菌DNA为模板扩增出特异的PCR产物。此外，该方法灵敏度高，检测模板浓度可低至0.35 pg/μL。构建的荧光定量PCR标准曲线Ct值与模板浓度呈良好的线性关系，熔解曲线的吸收峰单一，扩增效率好。利用该定量检测体系，能从几种不同的三七病株中快速检测出携带尖孢镰刀菌的根腐病病株，从而达到准确诊断的目的。本方法不仅能明确三七种植土壤以及三七病株中尖孢镰刀菌数量的动态变化，并且可以为三七土壤处理、根腐病的早期诊断和动态监测以及带病三七种子、种苗的快速分子检测提供技术支持。

关键词: 三七根腐病, 尖孢镰刀菌, 分子检测, 实时荧光定量PCR, 快速检测

Abstract: The aim of this paper is to establish an accurate and rapid Real-time fluorescence quantitative PCR method for the detection of Fusarium oxysporum, the pathogenic fungus of Panax notoginseng root rot. A pair of specific primer, Fo-QF and Fo-QR, was designed based on the fatty acid ω-hydroxylase gene of F.oxysporum in this study. The recombinant plasmid of this gene was prepared, moreover, the SYBR Green Ⅰ fluorescence Real-time quantitative PCR method for detecting F. oxysporum was established. In addition, the 14 samples including P. otoginseng plants with typical symptoms of root rot and black spot as well as the P. notoginseng planting soil were collected. The total DNAs of these samples were extracted as templates and detected. The results showed that the Real-time fluorescence quantitative PCR method established was specific. The fatty acid ω-hydroxylase gene could amplify specific PCR products only when F. oxysporum DNA was used as templates. In addition, the method had high sensitive, and the concentration of detection template could be as low as 0.35 pg/μL. The Ct of the standard curve constructed had a good linear relationship with the centrations of templates. The absorption peak of the dissolution curve was single, and the amplification efficiency was good. With the quantitative detection system of this study, the P. notoginseng root rot plants with F. oxysporum were rapidly detected from several different P. notoginseng disease strains. So it could achieve the purpose of accurate diagnosis. This method can be used to reveal the dynamic changes of F. oxysporum numbers in the complex P. notoginseng planting soil and in the 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. notoginsen g seeds and seedlings.

Key words: Panax notoginseng root rot, Fusarium oxysporum, Molecular detection, Real-time PCR, Rapid detection

中图分类号:

S432.4

李欣, 崔秀明, 刘迪秋, 孙恬, 郭爱玲, 陈军. 三七根腐病菌尖孢镰刀菌的Real-time PCR检测方法[J]. 华北农学报, 2019, 34(S1): 324-330. doi: 10.7668/hbnxb.20190370.

LI Xin, CUI Xiuming, LIU Diqiu, SUN Tian, GUO Ailing, CHEN Jun. Real-time PCR Detection Method of Panax notoginseng Root Rot Pathogen Fusarium oxysporum[J]. ACTA AGRICULTURAE BOREALI-SINICA, 2019, 34(S1): 324-330. doi: 10.7668/hbnxb.20190370.

http://www.hbnxb.net/CN/Y2019/V34/IS1/324

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