葡萄根瘤蚜TaqMan-MGB实时荧光定量检测方法的研究

Detection of grape phylloxera by real-time fluorescent PCR assay using TaqMan-MGB probes

为建立葡萄根瘤蚜实时荧光定量PCR的检测方法,参考Karen Herbert等设计的特异性引物与TaqMan-MGB荧光探针,构建以标准阳性质粒作为标准品制作标准曲线,并经优化反应条件,建立葡萄根瘤蚜的实时荧光PCR绝对定量检测方法,进行敏感性和重复性试验,并对受葡萄根瘤蚜为害的葡萄根际土壤进行初步定性检测。结果表明:该方法的灵敏度可达1.625拷贝/μL,3次重复检测的变异系数均小于5%。提取0.25g含有10头葡萄根瘤蚜若虫土壤的DNA,并将其梯度稀释,用建立的荧光定量PCR进行检测,将DNA稀释103倍后,仍能检测出阳性结果。对受害葡萄根际土壤检测结果为阳性。葡萄根瘤蚜TaqMan-MGB探针实时荧光PCR检测技术具有特异性强,敏感性高,易操作等优点,有很好的应用前景和研究价值。

According to real-time PCR method for detection of grape phylloxera,established with specific primers and TaqMan-MGB probe by Karen Herbert et al,the recombinant plasmid containing the ITS2 sequence was constructed as a standard control,and then an absolute quantitative method of real-time PCR was developed by optimizing reaction conditions,and sensitivity and reproducibility were verified,and rhizosphere soil of grape infested by phylloxera was detected.The sensitivity of this method was proved to be 1.625 copies/μL,and the coefficient of variation was less than 5%.The DNA,distilled from 0.25 g soil containing 10 grape phylloxera crawlers,could be detected efficiently,even diluted a thousand times.Rhizosphere soil was detected positive.The TaqMan-MGB probe-based real-time fluorescent PCR assay served as a promising approach for easy,rapid and highly specific and sensitive detection of grape phylloxera.