南方农业学报2012，Vol.43Issue(5)：616-620,5.DOI:10.3969/j:issn.2095-1191.2012.05.616
甘蔗宿根矮化病PCR检测技术优化分析
Optimization of PCR technique for detecting sugarcane ratoon stunting disease
摘要
Abstract
[Objective]Based on the basic procedure of PCR, the present study used specific primers mapped from 16S-23S rDNA of Leifsonia xyli subsp. xyli (Lxx), optimized the PCR reaction conditions, to establish a stable, fast and highly sensitive PCR detection technique for sugarcane ratoon stunting disease. [Method]Using Lxx genomic DNA as templates, Ex Taq enzyme (TaKaRa) and 2xGC Buffer Ⅱ , the present study adjusted the main conditions such as the annealing temperature and the concentration of primers etc. to optimize the PCR reaction system; detected the sensitivity of optimized PCR with diluted Lxx genomic DNA templates; and used the optimized PCR reaction system to determine the RSD of total DNA of the leaves from sugarcane cultivar GT11 growing in the field. [Result]By the optimized PCR detection system, the clear and specific band for internal transcribed spacer of Lxx 16S-23S rDNA was obtained. The optimized PCR detection system could detect Lxx from the 1000 times diluted Lxx genomic DNA (15.9 pg/μL), while the common PCR detection system could only do it from the 100 times diluted Lxx genomic DNA (159 pg/μL), and also produce false negativeness with unstable results. The optimized PCR detection system was verified by detecting the Lxx from the leaves of sugarcane cultivar CT11 growing in the field, which showed 66.7% RSD infection rate, while 0 RSD infection rate was detected by the common PCR detection system. [ Conclusion ]The optimized PCR detection system was listed below: 2×GC buffer Ⅱ 12.5 μL,Ex Tag DNA enzyme (5U/μL) 0.2 μL;dNTP (2.5 mmol/LH.5 μL：Lxxl (10 μmol/L) 1.0 μL,Lxx2 (10 μmol/L) 1.0 μL.DNA template 1.0 μL,and add ddH2O to complement the total volume of 25.0 μL. PCR reaction procedure was like this:initial denaturated 10 min at 95℃,15 s at 94℃,15 s at 57℃, 30 s at 72℃. 40 recycles;then extended 10 min at 72℃. The optimized PCR detection system was confirmed more sensitive, stable and efficient than the common PCR detection system, so it is more desirable for rapid detection of RSD from mass field samples.关键词
甘蔗/宿根矮化病/PCR检测/16S～23S rDNA/优化体系Key words
sugarcane/ raloon stunting disease/ PCR detection/ 16S-23 S rDNA/ optimized system分类
农业科技引用本文复制引用
周丹,谢晓娜,陈明辉,杨丽涛,李杨瑞..甘蔗宿根矮化病PCR检测技术优化分析[J].南方农业学报,2012,43(5):616-620,5.基金项目
科技部国际合作项目(2008DFA30600,2009DFA30820) （2008DFA30600,2009DFA30820）
广西自然科学基金创新研究团队项目(2011GXNSFF018002) （2011GXNSFF018002）
广西农业科学院创新研究团队项目(桂农科2011YT01) （桂农科2011YT01）
