番茄褪绿病毒SYBR Green I实时荧光定量PCR方法
SYBR Green I quantitative real-time PCR ( qPCR) for Tomato chlorosis virus
查看参考文献30篇
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文摘
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根据番茄褪绿病毒( Tomato chlorosis virus,ToCV)热激蛋白70( Hsp70)的基因序列,设计ToCV实时荧光定量PCR特异引物.利用重组质粒ToCV-1为标准品建立SYBR Green I实时荧光定量方法.针对引物浓度、退火温度、特异性、灵敏度、重复性和稳定性进行系列优化.结果表明,最适退火温度为63℃,最适引物浓度为0.3 μmol·L~(-1).熔解曲线为特异性单峰,表明其特异性良好.建立的SYBR Green I实时荧光定量PCR较常规PCR灵敏100倍,且具有良好的重复性和稳定性.基于SYBR Green I实时荧光定量PCR技术建立的ToCV检测方法,速度快、特异性强、灵敏度高、重复性好,可以用于ToCV的定量检测. |
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其他语种文摘
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According to the heat shock protein 70 ( Hsp70) gene sequence of Tomato chlorosis virus ( ToCV) , a specific primer pair for ToCV real-time fluorescent quantitative PCR was designed. The recombinant plasmid ToCV-1 is used as a standard to establish a SYBR Green I real-time fluorescence quantitative method and a series of optimization include primers concentration,annealing temperature,specificity,sensitivity,reproducibility and stability were performed. The results showed that,the optimized annealing temperature is 63 ℃,and the primer concentration is 0.3 μmol·L~(-1). The melting curve for specific peak proved that this method has good specificity. By comparing with the sensitivity of conventional PCR,we found that the SYBR Green I real-time fluorescence quantitative PCR was 100 times more sensitive. The method has good repeatability and stability. The rapid detection method based on SYBR Green I real-time quantitative PCR has high speed,strong specificity, high sensiti-vity and good repeatability. It can be used for the quantitative detection of ToCV. |
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来源
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植物病理学报
,2018,48(5):700-706 【核心库】
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DOI
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10.13926/j.cnki.apps.000230
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关键词
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番茄褪绿病毒
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SYBR Green I实时荧光定量PCR
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病毒检测
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地址
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1.
山东农业大学植物保护学院, 山东省蔬菜病虫生物学重点实验室;;山东果蔬优质高效生产协同创新中心, 泰安, 271018
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荣成市港湾街道办事处, 荣成, 264309
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潍坊科技学院, 寿光, 262700
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语种
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中文 |
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文献类型
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研究性论文 |
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ISSN
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0412-0914 |
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学科
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植物保护 |
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基金
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山东省科技重大创新工程
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山东省自然科学基金
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潍坊市科学技术发展计划项目
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文献收藏号
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CSCD:6364853
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