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

作　　者：孙晓辉[1] 高利利 刘锦[1] 王少立[1] 乔宁[1,3] 刘永光[3] 赵静[3] 竺晓平[1] SUN Xiao-hui1 , GAO Li-li2, LIU Jin1, WANG Shao-li1, QIAO Ning1,3, LIU Yong-guang3, ZHAO Jing3 , ZHU Xiao-ping1(1 College of Plant Protection, Shandong Agricultural University, Shandong Provincial Key Laboratory for Biology of Vegetable Diseases and Insect Pests, Collaborative Innovation Center of Fruit ＆ Vegetable Quality and Efficient Production in Shandong, Taian 271018, China; 2Rongcheng City Harbor Subdistrict Office, Rongcheng 264309, China; 3Weifang University of Science and Technology, Shouguang 262700, China)

机构地区：[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 [2]荣成市港湾街道办事处,荣成264309 [3]潍坊科技学院,寿光262700

出　　处：《植物病理学报》2018年第5期700-706,共7页Acta Phytopathologica Sinica

基　　金：山东省科技重大创新工程(2017CXGC0207);山东省自然科学基金(ZR2017MC061);潍坊市科学技术发展计划项目(2017GX076)

摘　　要：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0.3μmol·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。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 sensitivity and good repeatability. It can be used for the quantitative detection of ToCV.

关 键 词：番茄褪绿病毒 SYBR Green I实时荧光定量PCR 病毒检测 

分 类 号：S432.41[农业科学—植物病理学]