棉花黄萎病菌实时荧光定量PCR检测方法的建立及应用

Development and application of a real-time PCR assay for the detection and quantification of Verticillium dahliae

利用棉花黄萎病菌ITS区特异性引物建立了棉花黄萎病菌的实时荧光定量PCR检测方法。利用实时荧光PCR体系,以324 bp的PCR扩增产物构建了标准曲线并对该方法的灵敏度、特异性和重复性进行了评估。结果表明:该方法特异性良好,检测灵敏度为100 copies·μL-1,标准曲线的相关系数为0.994,扩增效率为91.5%。利用建立的检测方法对转基因棉田及常规棉田土壤样本进行检测,结果表明转基因棉田中棉花黄萎病菌数量显著高于常规棉田,与实际观测到的现象一致,也证明了本方法的可行性。因此,本研究建立的棉花黄萎病菌检测方法具有灵敏度高、重复性好等特点,为棉花的种植及病害防治提供了有效的检测手段。

A specific real-time PCR assay based on the ITS region of rDNA was established to quantitatively detect Verticillium dahliae of cotton in a naturally infested soil. The standard curve of the real-time PCR system was constructed by a 324 bp PCR amplification product and the spe- cificity, sensitivity and reproducibility of the method were evaluated. The results showed that the real-time PCR assay had good specificity and sensitivity and the detection limit was 100 copies · μL-1. The correlation coefficient of the standard curve was 0.994 and the PCR amplification effi- ciency reached 91.5%. Using the real-time PCR assay, soil samples from a transgenic cotton field and nontransgenie one were detected. The results indicated that the amount of V. dahliae in the transgenic cotton field was significantly higher than that of conventional cotton, which showed consistency with the observations achieved in the two cotton fields. As a reliable and sensitive as- say, the real-time PCR system provided an effective method for the rapid detection of V. dahliae and the control of cotton diseases.