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.%建兰花叶病毒(Cymbidium mosaic virus,CyMV)是引起兰科植物病毒病的最重要病原体.研究根据NCBI上已登录的CyMV外壳蛋白(Coat Protein,CP)基因保守区设计两对特异性引物,用RT-PCR法从感病蝴蝶兰中克隆到2条CyMV CP基因序列,编码区ORF长672 bp,编码223个氨基酸.序列分析表明这两个分离物之间有13个核苷酸差异,与已报道的其他地区CyMV分离物CP基因核苷酸相似性分别为97%~99%和97%~98%,氨基酸相似性分别为95%~99%和96%~100%.运用实时荧光定量RT-qPCR法对该病毒检测方法进行了一系列条件优化,建立起一套基于SYBR Green I的CyMV实时荧光定量PCR检测体系.该检测方法比普通RT-PCR法灵敏度高100倍,标准曲线循环阈值(Ct)与模板浓度的对数呈现良好的线性关系,扩增效率为100%,相关性系数为0.998.重复性试验表明组内及组间变异系数均在2.58%以内,表明该方法重复性较好,可为兰花CyMV的早期预警及病毒在植物体内侵染途径研究提供技术支持.
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