中国农业科学Issue(1)：55-62,8.DOI:10.3864/j.issn.0578-1752.2015.01.06
SYBR GreenI实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用
Establishment of SYBR Green I Real-Time PCR for Quantitatively Detecting Rhizoctonia cerealis in Winter Wheat
摘要
Abstract
[Objective]Wheat sharp eyespot (WSE) caused by Rhizoctonia cerealis is one of the most important soil-born diseases on wheat in China. Early accurate quantitative detection is a foundation of forecast and control. Traditional method of organization isolation and identification of pathogen is time consuming, complicated and can’t be accurately quantified. In order to implement the early and quick quantitative determination of wheat sharp eyespot, a SYBR Green I real-time PCR method of R. cerealis was established based on the pathogen sequence information. [Method] Based on the β-tubilin of R. cerealis, a pair of specific primers was designed. The SYBR Green I real-time PCR reaction system was established and optimized. The sensitivity, specificity and repeatability of the system were also evaluated, and R. cerealis, R. solani, AG-A, AG-F, Gaeumannomyces graminis var. tritici, Bipolaris sorokiniana, Fusarium graminearum, F. pseudograminearum were used for control fungi. The indoor potted plants of wheat which infected by R. cerealis were detected with optimized reaction system after inoculated for 5, 10 and 60 days, respectively.[Result]The primers were of great specificity, the specific PCR fragment was amplified from the DNA of R. cerealis isolates, but not from the DNA of other fungal isolates by conventional PCR. The real-time PCR assays also did not amplify DNA from control fungi. The sensitivity of conventional PCR was 6.5×103 copies/μL plasmid, while the sensitivity of real-time PCR was 6.5×102 copies/μL. The standard curve established by recombinant plasmid showed a fine linear relationship between threshold cycle and template concentration. The melt curve was specific with the correlation coefficient of 0.997 and with high amplification efficiency (0.91). For the indoor potted experiments, the detection results of real-time PCR of infected wheat samples, were showed a significant positive correlation with disease index and inoculum, respectively.[Conclusion]The developed real-time PCR assay for R. cerealis is fast, highly specific, sensitive, and reproducible. This method can be used to detect R. cerealis in wheat, and guidance prediction and control of wheat sharp eyespot.关键词
小麦纹枯病菌/SYBR Green I/实时荧光定量 PCR/早期检测Key words
Rhizoctonia cerealis/SYBR Green I/real-time PCR/early detection引用本文复制引用
孙炳剑,陈清清,袁虹霞,施艳,李洪连..SYBR GreenI实时荧光定量PCR检测小麦纹枯病菌体系的建立和应用[J].中国农业科学,2015,(1):55-62,8.基金项目
国家“十二五”粮食丰产科技工程（2012BAD04B07）、中国科学院知识创新工程重大项目 （）
