LNA探针实时荧光定量PCR检测小麦矮缩病毒体系的建立

 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0.5 μmol/L和0.3 μmol/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。

Detection of Wheat dwarf virus by TaqMan LNA probe real-time PCR

This study was conducted to develop a TaqMan Locked Nucleic Acid (LNA) probe real-time PCR assay that had a wide range of application, high sensitivity, and stability for the detection of Wheat dwarf vius (WDV) in the field. The specific primers and LNA probe (19 bp) were designed from conserved WDV-Rep sequences obtained from GenBank. The method was established to detect samples infected with WDV in the fields by TaqMan LNA probe real-time PCR. Matrix method was used to optimize the reaction system including the best concentration of the primers (0.5 μmol/L) and the probe (0.3 μmol/L). The slope and correlation coefficient of the standard curve were -3.424 3 and 0.997 3 , respectively, and the amplification efficiency was up to 96%. The coefficient of variation ranging from 0.11%-1.34% intra group and 0.44%-1.21% inter group indicated the excellent stability and reproducibility of the method. The sensitivity compared with the conventional PCR was analyzed by ten-fold dilution method. Results showed that the sensitivity of the former (55 copies/μL) was at least 100 times higher than the latter. In addition, the designment of probe become simpler and more convenient than TaqMan probe real-time PCR due to that the greatly shortened LNA probe is not prone to forming intermolecular and intramolecular secondary structures. Our results indicated that the detection method based on LNA probe is feasible for WDV early monitoring in the field, thus providing an efficient and rapid tool for WDV epidemiological investigation.