江西农业大学学报2017，Vol.39Issue(3)：572-580,9.DOI:10.13836/j.jjau.2017074
建兰花叶病毒SYBR Green I实时荧光定量PCR检测方法的建立
Development of a SYBR Green I Real-time Fluorescence Quantitative PCR Detection Method for Cymbidium mosaic virus
摘要
Abstract
Cymbidium mosaic virus(CyMV)is an important pathogen which causes widespread virus disease in orchid.In this study,two pairs of specific primers were designed according to the coat protein(CP)gene for CyMV on NCBI web.Two sequences of CyMV CP gene were obtained from the infected Phalaenopsis hybrid by RT-PCR,and the full-length was 672 bp encoding 233 putative amino acid residues.Sequence analysis indicated that the two Zhengzhou isolates had 13 different nucleotides,and the sequences shared 97%-99% and 97%-98% similarities at nucleotides level and 95%-99% and 96%-100% at amino acid level with those of other CyMV strains.A series test conditions were optimized to establish an efficient real-time fluorescence quantitative PCR(RT-qPCR)detection method with SYBR Green I for CyMV.The results indicated that the detection sensitivity of RT-qPCR method was 100 times higher than that of regular PCR.Cycle threshold(Ct)and template concentration(log)showed a good linear relationship in the standard curve with 100% amplification efficiency and R2=0.998.Three-time repeat tests showed that the coefficients of variation between the intra-and inter-assay were both within 2.58%,indicating that the method had good repeatability and could be used to CyMV detection in orchid.关键词
建兰花叶病毒/实时荧光定量PCR/外壳蛋白基因/病毒检测Key words
Cymbidium mosaic virus/real-time fluorescence quantitative PCR/coat protein gene/virus detection分类
农业科技引用本文复制引用
梁芳,张燕,王若斓,许申平,程喜梅,崔波..建兰花叶病毒SYBR Green I实时荧光定量PCR检测方法的建立[J].江西农业大学学报,2017,39(3):572-580,9.基金项目
河南省科技攻关项目(162102110073)、郑州市科技攻关项目(141PPTGG420)和河南省产学研合作计划项目(152107000071)资助 Project supported by Henan Science and Technology Project (162102110073),Zhengzhou Science and Technology Project(141PPTGG420)and Henan Industry University Research Cooperation Project(152107000071) （162102110073）
