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| 甘薯褪绿矮化病毒西非株系实时荧光定量PCR检测方法的建立及应用 | |
| 引用本文： | 王丽,王振东,乔奇,秦艳红,张德胜,田雨婷,王爽,张立军,张振臣.甘薯褪绿矮化病毒西非株系实时荧光定量PCR检测方法的建立及应用[J].植物病理学报,2014,44(5):461-468. |
| 作者姓名： | 王丽 王振东 乔奇 秦艳红 张德胜 田雨婷 王爽 张立军 张振臣 |
| 作者单位： | 沈阳农业大学农学院,沈阳 110161; 河南省农业科学院植物保护研究所,河南省农作物病虫害防治重点实验室, 农业部华北南部作物有害生物综合治理重点实验室,郑州 450002; 南阳师范学院生命科学与技术学院,南阳 473061 |
| 基金项目： | 国家甘薯产业技术体系建设项目资助(CARS-11-B-07); 河南省农业科学院财政预算项目资助 |
| 摘 要： | 依据GenBank中登录的甘薯褪绿矮化病毒(Sweet potato chlorotic stunt virus,SPCSV)西非株系(WA)的核苷酸序列,分别设计两对特异性引物和一条TaqMan探针。以SPCSV-WA外壳蛋白(cp)基因的重组质粒为阳性标准质粒绘制标准曲线,通过优化反应体系和反应条件,建立了SPCSV-WA的实时荧光定量PCR检测方法。试验结果表明,该方法只能检测到目的病毒,标准曲线的斜率和相关系数分别为-3.239和1,扩增效率为103.568%。最低可检测到约3.31 copies/μL的阳性质粒,灵敏度比常规PCR高1 000倍。本研究建立的SPCSV实时荧光定量PCR方法可用于田间样品的检测,为SPCSV的早期预警和流行学研究提供了技术手段。 |
| 关 键 词： | 甘薯褪绿矮化病毒西非株系 荧光探针 实时荧光定量PCR |
| 收稿时间： | 2013-12-11 |
Development and application of a real-time PCR method for detection of west African strain of Sweet potato chlorotic stunt virus |
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| WANG Li,WANG Zhen-dong,QIAO Qi,QIN Yan-hong,ZHANG De-sheng,TIAN Yu-ting,WANG Shuang,ZHANG Li-jun,ZHANG Zhen-chen.Development and application of a real-time PCR method for detection of west African strain of Sweet potato chlorotic stunt virus[J].Acta Phytopathologica Sinica,2014,44(5):461-468. | |
| Authors: | WANG Li WANG Zhen-dong QIAO Qi QIN Yan-hong ZHANG De-sheng TIAN Yu-ting WANG Shuang ZHANG Li-jun ZHANG Zhen-chen |
| Institution: | College of Agronomy, Shenyang Agricultural University, Shenyang 110161, China; Institute of Plant Protection, Henan Academy of Agricultural Sciences, Henan Key Laboratory of Crop Pest Control, IPM Key Laboratory in Southern part of North China for Ministry of Agriculture, Zhengzhou 450002, China; Department of Life Sciences, Nanyang Normal University, Nanyan 473061,China |
| Abstract: | A sensitive and stable real-time PCR method for detecting west African strain of Sweet potato chlorotic stunt virus (SPCSV-WA) was established based on specific primers and TaqMan probe, which were designed from the coat protein gene sequences of SPCSV-WA. The standard curves was generated by purified DNA of the recombined plasmid of SPCSV-WA coat protein gene. With the recombined plasmid DNA as template, the main influential factors of real-time PCR including annealing temperature, probe and primer concentrations were optimized for the highest sensitivity. The results showed that the correlation coefficient and the slope value of the standard curves with plasmid DNA were 1 and -3.239, respectively. The efficiency of the PCR was 103.568%. The detection limit of the real-time PCR method was 3.31 copies/μL. Compared to conventional PCR, the real-time PCR method showed 1 000 fold higher in detection sensitivity. This method could be used for the specific detection of SPCSV-WA from field samples of sweet potato, which provided a useful tool for the early warning of SPCSV-WA and epidemiological investigation. |
| Keywords: | west African strain of Sweet potato chlorotic stunt virus (SPCSV-WA) TaqMan probe real-time PCR |
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