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本研究通过对多个小麦矮缩病毒(Wheat dwarf virus,WDV)小麦分离物进行序列分析,在编码Rep蛋白N端的基因保守区域设计一对特异性引物和一条长度为19 bp的TaqMan LNA探针,经过探针和引物浓度的系列优化,建立了WDV田间样品LNA探针实时荧光定量PCR检测方法,确定最优反应条件下的引物和探针终浓度分别为0.5μmol/L和0.3μmol/L。该方法建立的标准曲线斜率及相关系数分别为-3.424 3和0.997 3,扩增效率为96.0%,重复性试验组内变异系数为0.11%~1.34%,组间变异系数为0.44%~1.21%,最低检测限为55 copies/μL,其灵敏度是普通PCR的100倍以上。与普通TaqMan探针荧光定量PCR相比,该方法所用探针长度大大缩短,减少了探针间形成二级结构的机会,探针设计更加简单、方便。该方法适用于田间样品的早期监测,为病害流行调查提供了高效快捷的技术支撑。
In this study, a series of specific primers and a 19 bp TaqMan LNA probe were designed by sequence analysis of several wheat dwarf virus (WDV) wheat isolates in the conserved region of the gene encoding the N-terminus of Rep protein. . After real-time fluorescence quantitative PCR detection of LNA probe in WDV field samples, the optimized primers and probe final concentrations were 0.5μmol / L and 0.3μmol / L . The slope of the standard curve and the correlation coefficient established by this method were -3.424 3 and 0.997 3 respectively, the amplification efficiency was 96.0%, the coefficient of variation in the repeated test group was 0.11% -1.34%, and the coefficient of variation among the groups was 0.44% -1.21% , The minimum detection limit of 55 copies / μL, its sensitivity is 100 times more than normal PCR. Compared with the ordinary TaqMan probe fluorescence quantitative PCR, the length of the probe used in the method is greatly shortened, thereby reducing the chance of forming secondary structure between the probes, and the probe design is simple and convenient. The method is suitable for the early monitoring of field samples and provides efficient and quick technical support for the epidemic investigation of diseases.