甜瓜黄斑病毒SYBR Green Ⅰ实时荧光定量PCR方法  

SYBR Green Ⅰ quantitative real-time PCR for melon yellow spot virus

作　　者：孙晓辉[1] 潘睿婧 刘永光[2] 王丹 石朝鹏 乔宁[2] 孙作文 竺晓平[1] SUN Xiaohui;PAN Ruijing;LIU Yongguang;WANG Dan;SHI Zhaopeng;QIAO Ning;SUN Zuowen;ZHU Xiaoping(College of Plant Protection,Shandong Agricultural University,Collaborative Innovation Center of Fruit and Vegetable Quality and Efficient Production in Shandong,Tai′an 271018,China;Weifang University of Science and Technology,Shandong Facility Horticulture Bioengineering Research Center,Shouguang 262700,China;Shandong Provincial Plant Protection Central Station,Jinan 250100,China)

机构地区：[1]山东农业大学植物保护学院,山东省果蔬优质高效生产协同创新中心,泰安271018 [2]潍坊科技学院,山东省设施园艺生物工程研究中心,寿光262700 [3]山东省植物保护总站,济南250100

出　　处：《植物病理学报》2023年第1期119-125,共7页Acta Phytopathologica Sinica

基　　金：山东省农业重大应用技术创新项目(SD2019ZZ004);山东省重大科技创新工程(2019JZZY010707);山东省自然科学基金(ZR2017MC061)。

摘　　要：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0.65μmol·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。The aim of this study was to establish a SYBR Green Ⅰ quantitative real-time PCR method(qPCR) for detection of melon yellow spot virus(MYSV). The specific primer pair for qPCR was designed based on the conserved sequence of MYSV nucleocapsid protein gene, and the annealing temperature, primer concentration, specificity and sensitivity of the primer were optimized. The results showed that the optimal annealing temperature of the optimized qPCR method was 61.3 ℃, the optimal primer concentration was 0.65 μmol·L-1, the specificity and repeatability were good and this method was 100 times more sensitive than PCR. The cycle threshold of the qPCR standard curve was linear with the template concentration, and the correlation coefficient was 0.999 7. The results of field samples showed that the established qPCR method can be used for the quantitative detection of MYSV.

关 键 词：甜瓜黄斑病毒 SYBR GreenⅠ 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]