| 谢锦添,俞超,谭志文,吴月燕,马立孟.猕猴桃溃疡病菌实时荧光定量PCR检测方法的建立和应用[J].中国南方果树,2017,46(6): |
| 猕猴桃溃疡病菌实时荧光定量PCR检测方法的建立和应用 |
| Establishment and application of real－time PCR method for detection of Pseudomonas syringae pv. Actinidae |
| 投稿时间：2017-07-17 修订日期：2017-09-08 |
| DOI： |
| 中文关键词: 猕猴桃溃疡病菌 实时荧光定量PCR 检测方法 应用 |
| 英文关键词:Pseudomonas syringae pv. Actinidae real‐time PCR detection method application |
| 基金项目: |
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| 摘要点击次数: 1850 |
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| 中文摘要: |
| 为建立猕猴桃溃疡病病原菌PSA的实时荧光定量PCR方法，根据hrpW基因设计一对引物P3F/P5R，扩增片段大小为247bp，构建该片段的阳性质粒用于荧光定量 PCR标准曲线的制作；评价引物的灵敏度、特异性，并验证其实际应用效果。结果表明，该方法能特异性的检测出PSA病原菌，灵敏度为 100fg/μL。以采集的无病症和有病症猕猴桃枝条为样品，本方法可检测到无病症枝条中最低浓度为8.45?0‐5ng/μL的PSA。本实验建立的实时荧光定量PCR检测猕猴桃溃疡病菌方法可用于猕猴桃溃疡病感病样品的早期诊断，对该病原菌的预防诊治具有重要意义。 |
| 英文摘要: |
| To establish a real-time PCR method of pathogenic bacteria （Pseudomonas．Syringae pv．Actinidae，PSA）detection , the pair of primers P3F/P5R were screened out according to hrpW gene sequence, with expected amplicons of 247bp in length, were used in real-time PCR. The recombinant plasmid of this amplification were used to build a standard curve real-time PCR . The sensitivity specificity of these primers were analyzed and its real effect was validated. The results of sensitivity assays showed the sensitivity threshold of the real-time PCR was 100fg/μL. The results of actual effect validation experiments showed part of symptomless samples were positively detected by real-time PCR, with minimum DNA concentration 8.45?0‐5ng/μL. The methods in this study could be expected to greatly contribute to the early diagnosis and control of the bacterial canker disease on kiwifruit. |
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