棉花黄萎病菌实时荧光定量PCR检测方法的建立及应用

生态学杂志

棉花黄萎病菌实时荧光定量PCR检测方法的建立及应用

沈元劼¹,齐谢敏²,刘标³,周国华^1,2,李泰明^1**

(¹中国药科大学生命科学与技术学院，南京 210009； ^2南京大学医学院附属金陵医院，南京 210093； ³国家环保总局南京环境科学研究所，南京 210037)

出版日期:2015-07-10 发布日期:2015-07-10

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

SHEN Yuan-jie¹, QI Xie-min², LIU Biao³, ZHOU Guo-hua^1,2, LI Tai-ming^1**

(¹School of Life Science and Technology, China Pharmaceutical University, Nanjing 210009, China; ²Jingling Hospital, Medical School of Nanjing University, Nanjing 210093, China; ³Nanjing Institute of Environmental Sciences, Ministry of Environmental Protection, Nanjing 210037, China)

Online:2015-07-10 Published:2015-07-10

摘要/Abstract

摘要： 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。

关键词: 水文频率, 水文基因, 进化原理, 常规矩, 非一致性

Abstract: 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 specificity, 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 efficiency reached 91.5%. Using the realtime PCR assay, soil samples from a transgenic cotton field and nontransgenic 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 assay, the realtime PCR system provided an effective method for the rapid detection of V. dahliae and the control of cotton diseases.

Key words: evolution principle, hydrologicalfrequency., moment, non-consistency, hydrological gene

沈元劼1,齐谢敏2,刘标3,周国华1,2,李泰明1. 棉花黄萎病菌实时荧光定量PCR检测方法的建立及应用[J]. 生态学杂志.

SHEN Yuan-jie1, QI Xie-min2, LIU Biao3, ZHOU Guo-hua1,2, LI Tai-ming1**. Development and application of a real-time PCR assay for the detection and quantification of Verticillium dahliae.[J]. cje.