应用Taqman探针实时荧光定量PCR技术检测烟草靶斑病菌Rhizoctonia solani AG-3方法的建立

Quantitative detection of Rhizoctonia solani AG-3 in tobacco by Taqman real-time PCR

分别基于7个烟草靶斑病菌株基因组中ITS-5.8S r DNA序列设计引物和探针;并对引物及探针特异性进行验证;建立检测体系并对接种烟草靶斑病菌的叶片和土壤中的烟草靶斑病菌进行检测。结果表明:所设计的引物及探针对R.solani AG-3具有特异性,检测体系可以检测出烟草叶片及土壤样品中的烟草靶斑病菌。接种烟草叶片的检测表明,接种后6h就可检测到强致病力菌株YC-9,12h后能检测到弱致病力菌株LF-2;获得了烟草靶斑病菌DNA质量的对数与添加菌丝量的对数之间的回归曲线方程,对不同月份土壤样品的测定结果表明烟草靶斑病菌在土壤中呈周年动态变化趋势。

A pair of primers and probe was designed based on ITS- 5.8 S r DNA sequences of 7 strains R. solani AG-3 and specificity was certificated. Detection system was established to detect DNA content of tobacco target spot pathogens from tobacco leaves and soil. Results showed that the designed probes and primers were available to detect R. solani AG-3 from tobacco leaf and soil samples. Strong virulent strains YC-9 could be detected 6h after inoculation, while weak virulent strains LF-2 could be detected 12 h after inoculation. Regression curve equation between logarithmic of DNA copy number and logarithmic of adding amount of hypha for tobacco target spot pathogen was established. Detection of soil samples of different months showed that tobacco target spot pathogen had an annual dynamic trend in soil.