TaqMan探针双重实时荧光定量PCR法同步检测兰花中的建兰花叶病毒和齿兰环斑病毒
Simultaneous detection of cymbidium mosaic virus and odontoglossum ringspot virus by TaqMan duplex real-time fluorescence quantitative PCR
孙爱青 1王丽花 2张艺萍 2杨秀梅 2许凤 2苏艳2
作者信息
- 1. 云南省农业科学院花卉研究所,云南省花卉育种重点实验室,国家观赏园艺工程技术研究中心,昆明 650205;云南大学,昆明 650091
- 2. 云南省农业科学院花卉研究所,云南省花卉育种重点实验室,国家观赏园艺工程技术研究中心,昆明 650205
- 折叠
摘要
为建立一种同步定量检测建兰花叶病毒(cymbidium mosaic virus,CymMV)和齿兰环斑病毒(odontoglossum ringspot virus,ORSV)的高效检测方法,本研究根据CymMV和ORSV CP基因高度保守区分别设计引物和探针并筛选,获得156 bp和148 bp的靶标序列及对应的最优特异性引物探针组合,建立了基于TaqMan探针的双重实时荧光定量PCR方法.该方法最低检出限为1拷贝或6.2×10-3fg,最低稳定检出限为10拷贝或6.2×10-2 fg,是RT-PCR的10～100倍;构建的标准曲线线性关系良好,扩增效率分别为97.7％和100.2％,相关系数R2 分别为1.000和0.999;对其他5种常见病毒均无扩增曲线,检测特异性强;批组内与批组间重复性试验Ct值变异系数≤0.60％,重复性和稳定性好.利用该方法和基因芯片法分别对4个种属的66个兰花样品进行方法验证,该方法相较于基因芯片法检出率提高了 53.85％(CymMV)和162.5％(ORSV).综上所述,本方法可同时高通量检测CymMV和ORSV 2种病毒靶基因,结果可靠,应用前景广阔,可为开展病毒精准鉴定、科学防控以及从源头遏制病毒传播提供技术支撑.
Abstract
To establish an efficient method for synchronous and quantitative detection of cymbidium mosaic virus(CymMV)and odontoglossum ringspot virus(ORSV),primers and probes were designed and screened according to the highly conserved regions of CymMV and ORSV CP genes.156 bp and 148 bp target sequences and the corresponding optimal specific primers and probe were obtained,and the TaqMan duplex real-time fluorescent quantitative PCR were established.The minimum detection limit was 1 copy or 6.2×10-3 fg.The minimum stable detection limit was 10 copies or 6.2×10-2 fg,whose sensitivity is 10-100 times as much as that of RT-PCR.The standard curve showed a good linear relationship with the amplification efficiencies of 97.7％and 100.2％,respectively,and the correlation coefficient R2 of 1.000 and 0.999,respectively.There was no amplification curve for the other five common viruses,indicating the method was very specific.The intra-and interassay CVs of Ct values was less than 0.60％,indicating the method was of good repeatability.In addition,66 leaves from four different orchid genera were analyzed using the established method and gene chip assay,and the detection rate of this method was 53.85％(CymMV)and 162.5％(ORSV)higher than that of gene chip assay,respectively.In conclusion,the TaqMan duplex assay established in this study can detect CymMV and ORSV simultaneously with reliable results,which has a board prospect of application and can provide technical support for accurate virus identification,scientific prevention and control,and containment of virus transmission from the source.
关键词
建兰花叶病毒/齿兰环斑病毒/TaqMan探针/RT-qPCR/兰花Key words
cymbidium mosaic virus(CymMV)/odontoglossum ringspot virus(ORSV)/TaqMan probe/qRT-PCR/orchid引用本文复制引用
基金项目
云南省种子种业联合实验室项目(202205AR070001-05)
云南省标准化研究项目(2023BZHXM05)
省级重大专项科技计划(农业)(202102AE090052)
出版年
2024
NETL
NSTL
万方数据