同步检测PMWaV-1、PMWaV-2和PMWaV-3的三重RT-qPCR方法的初步建立

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-qPCR)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TaqMan水解探针,在优化PMWaV-1、PMWaV-2、PMWaV-3三重RT-qPCR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-qPCR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-qPCR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-qPCR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.%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.