甘蔗宿根矮化病PCR检测技术优化分析  被引量:13

Optimization of PCR technique for detecting sugarcane ratoon stunting disease

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作  者:周丹[1,2] 谢晓娜[1,2] 陈明辉[1,2] 杨丽涛[1,2,3] 李杨瑞[1,2,3,4] 

机构地区:[1]广西大学农学院,南宁530005 [2]亚热带农业生物资源保护与利用国家重点实验室,南宁530005 [3]中国农业科学院甘蔗研究中心/农业部广西甘蔗生物技术与遗传改良重点实验室/广西甘蔗遗传改良重点实验室.南宁530007 [4]广西农业科学院广西作物遗传改良生物技术重点开放实验室,南宁530007

出  处:《南方农业学报》2012年第5期616-620,共5页Journal of Southern Agriculture

基  金:科技部国际合作项目(2008DFA30600,2009DFA30820);广西自然科学基金创新研究团队项目(2011GXNSFF018002);广西农业科学院创新研究团队项目(桂农科2011YT01)

摘  要:【目的】在PCR的基本程序上,以甘蔗宿根矮化病菌Lxx16S~23S rDNA内部转录间隔区特异性引物,优化PCR反应条件,以建立稳定、快速、灵敏度高的甘蔗宿根矮化病PCR检测技术。【方法】以Lxx基因组DNA为模板,采用TaKaRa的Ex Taq Polymerase和2×GCBufferⅡ,调整退火温度和引物浓度等主要因子,优化PCR反应体系;稀释Lxx基因组DNA模板的浓度,检测优化PCR的灵敏度;用优化的PCR对田间生长的甘蔗品种GT11叶片总DNA进行RSD测定。【结果】优化的PCR得到清晰特异的条带为Lxx16S~23S rDNA内部转录间隔区序列;能从稀释1000倍的Lxx基因组DNA(15.9pg/μL)中检测到Lxx,而常规的PCR只能从稀释100倍的Lxx基因组DNA(159pg/μL)中检测到Lxx,且出现假阴性,不稳定。对田间生长的甘蔗品种GT11叶片总DNA进行PCR检测,结果用优化后的PCR检测RSD感染率为66.7%,而常规的PCR检测RSD感染率为0。【结论】优化的PCR体系为:2×GCBufferⅡ12.5μL,Ex Taq DNA酶(5U/μL)0.2μL;dNTP(2.5mmol/L)1.5μL;Lxx1(10μmol/L)1.0μL,Lxx2(10μmol/L)1.0μL,DNA模板1.0μL,用ddH2O双蒸水补足至25.0μL。PCR反应程序为:95℃预变性10min,94℃15s,57℃15s,72℃30s,40个循环;72℃延伸10min。优化的PCR比常规PCR的灵敏度高、结果稳定、检测效率高,更适合于田间大批量样品快速检测。[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 geuomic DNA as templates, Ex Taq enzyme (TaKaRa) and 2×GC 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 GTll 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 cuhivar GT11 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 Taq DNA enzyme (5U/μL) 0.2 μL;dNTP (2.5 mmol/L)1.5 μL;Lxxl (10μmol/L) 1.0μL,Lxx2 (10 μmol/L) 1.0 μL,DNA template 1.0μL,and add ddH20 to complement the total volume of 25.0μL. 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

关 键 词:甘蔗 宿根矮化病 PCR检测 16S^23SrDNA 优化体系 

分 类 号:S435.661[农业科学—农业昆虫与害虫防治]

 

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