同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立 |
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引用本文: | 胡加谊,罗志文,何凡,李向宏,胡福初,范鸿雁,刘志昕,张治礼. 同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立[J]. 华北农学报, 2017, 32(2). DOI: 10.7668/hbnxb.2017.02.003 |
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作者姓名: | 胡加谊 罗志文 何凡 李向宏 胡福初 范鸿雁 刘志昕 张治礼 |
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作者单位: | 1. 海南省农业科学院 热带果树研究所,农业部海口热带果树科学观测实验站,海南省热带果树生物学重点实验室, 海南 海口 571100;黄埔出入境检验检疫局,广东 广州 510730;2. 海南省农业科学院 热带果树研究所,农业部海口热带果树科学观测实验站,海南省热带果树生物学重点实验室, 海南 海口 571100;3. 中国热带农业科学院 热带生物技术研究所,海南 海口,571101 |
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基金项目: | 海南省重点研发项目,公益性行业(农业)科研专项经费项目,国家自然科学基金项目 |
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摘 要: | 目前我国菠萝产区已相继报道3种菠萝凋萎相关病毒(PMWaV-1、PMWaV-2和PMWaV-3)。通过建立这3种菠萝凋萎相关病毒实时荧光定量RT-PCR(RT-q PCR)同步定量检测方法,为菠萝凋萎病毒的定性定量研究提供技术手段。根据这3种菠萝凋萎相关病毒的外壳蛋白基因序列分别设计特异性引物和Taq Man水解探针,在优化PMWaV-1、PMWaV-2、PMWaV-3三重RT-q PCR反应体系后,以PMWaV-1、PMWaV-2、PMWaV-3 CP基因目的片段的重组质粒为标准品绘制标准曲线,初步建立了PMWaV-1、PMWaV-2、PMWaV-3三重RT-q PCR检测方法。其中PMWaV-1标准曲线为y=-3.283logx+39.02,其扩增效率达101.7%,线性相关系数R2=0.999;PMWaV-2标准曲线为y=-3.393logx+37.53,其扩增效率为97.1%,线性相关系数R2=0.998;PMWaV-3标准曲线为y=-3.171 logx+38.48,其扩增效率为106.7%,线性相关系数R2=0.996;这表明3种菠萝凋萎相关病毒质粒标准品在同一反应体系中可稳定扩增,且线性关系表现良好,可用于后续试验。灵敏度试验数据表明,该方法对PMWaV-1、PMWaV-2、PMWaV-3的最低检测线分别为99,98,868拷贝;重复性试验表明PMWaV-1、PMWaV-2、PMWaV-3三重RT-q PCR扩增曲线的标准差小于0.267,变异系数小于1.44%。这表明建立的三重RT-q PCR检测方法可快速、准确、稳定地定量检测PMWaV-1、PMWaV-2、PMWaV-3等3种病毒,为PMWaV的定性定量研究和复合侵染机制的探索提供了技术基础。
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关 键 词: | 菠萝凋萎病 检测方法 实时荧光定量RT-PCR PMWaV TaqMan探针 |
Establishment of a Triple RT-qPCR Method for the Simultaneous Detecting of PMWaV-1,PMWaV-2 and PMWaV-3 |
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Abstract: | The objective of this paper was to provide a technique for quantitative and quanlitative studies of PMWaV by establishing the method of Real-time Fluorescent Quantitative RT-PCR(RT-qPCR)for simultaneous quantitative detection of PMWaV-1,PMWaV-2 and PMWaV-3.Primers and probes were designed and synthesized according to the CP gene of PMWaV-1,PMWaV-2 and PMWaV-3,respectively.After optimized the reaction system of triple RT-qPCR,the standard curves were generated by purified DNA of the recombined plasmid of PMWaV-1,PMWaV-2,PMWaV-3 coat protein gene,respectively.And the method of RT-qPCR for the simultaneous detection of PMWaV-1,PMWaV-2,PMWaV-3 had been improved.The amplification efficiency of PMWaV-1,PMWaV-2,PMWaV-3 in the RT-qPCR method was up to 101.7%,97.1%,106.7%,respectively.Meanwhile ,with the R2 between 0.996 and 0.999.The sensibility test of triple RT-qPCR syetem showed the detection limit of quantification for PMWaV-1,PMWaV-2,PMWaV-3 was 99,98 and 868 copies,respectively.Moreover,repeats experiment datas showed that standard deviation was below 0.26 and variable coefficient was below 1.44%.This study indicated that the method for simultaneous detection and quantification of PMWaV-1,PMWaV-2,PMWaV-3 was a fast,sably and sensitive reaction system,which could be the technological base for quantitative and quanlitative studies of PMWaV. |
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Keywords: | Mealybug Wilt of Pineapple Dctection method RT-qPCR PMWaV TaqMan probe |
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