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甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法

SUN Xiaohui, PAN Ruijing,LIU Yongguang,WANG Dan,SHI Zhaopeng,QIAO Ning,SUN Zuowen,ZHU Xiaoping

Acta Phytopathologica Sinica（2023）

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Abstract
为建立检测甜瓜黄斑病毒(melon yellow spot virus,MYSV)的SYBR Green Ⅰ实时荧光定量PCR(qPCR)方法.基于MYSV核衣壳蛋白基因保守序列设计qPCR特异性引物对,针对引物退火温度、引物浓度、特异性和敏感性进行系列优化.结果显示,优化后的qPCR方法最适退火温度为61.3℃,最适引物浓度为0.65 μmol·L-1,特异性强,灵敏度高,比PCR高100倍.以携带目的基因片段的重组质粒为标准品,构建的qPCR标准曲线循环阈值与模板浓度呈良好的线性关系,相关系数为0.999 7.实验样品验证表明建立的qPCR方法可用于MYSV的定量检测.
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Key words
melon yellow spot virus,SYBR Green Ⅰ,quantitative real-time PCR,virus detection
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