甘蔗宿根矮化病菌PCR检测体系的优化与应用  

Optimization and Application of PCR Detection for Leifsonia xyli subsp. xyli,the Causal Bacterium of Sugarcane Ratoon Stunting Disease

作　　者：李文凤[1] 卢文洁[1] 黄应昆 王明强[1] 王晓燕[1] 罗志明[1] 

机构地区：[1]云南省农业科学院甘蔗研究所云南省甘蔗遗传改良重点实验室,云南开远661600

出　　处：《云南农业大学学报》2011年第5期598-601,共4页Journal of Yunnan Agricultural University

基　　金：现代农业产业技术体系建设专项资金(CARS-20-2-2);公益性行业(农业)科研专项资金(ny-hyzx07-019);云南省人才培养项目(2008PY087)

摘　　要：为建立能快速灵敏检测甘蔗宿根矮化病(ratoon stuning disease,RSD)的PCR检测方法。本文通过改进模板DNA的制备方法,在PCR基本程序的基础上,以甘蔗宿根矮化病菌(Leifsonia xyli subsp.xyli,Lxx)16S-23SrDNA基因间隔区特异性引物,调整PCR扩增反应体系各组分的浓度、反应的退火温度、循环参数等主要因子,优化PCR反应体系。此检测体系能从感染RSD的样品中扩增出大小为438 bp的特异性片段,而阴性对照和未感染样品则未扩增出任何片断;未优化仅从稀释30倍的蔗汁总DNA中扩增出RSD的特异性片段,优化后可以从稀释3 000倍的蔗汁总DNA中扩增出RSD的特异性片段。对31份田间采集样品检测,未优化的阳性检出率为32.26%;优化后阳性检出率74.19%,与I-ELISA检测结果一致。应用此检测方法可稳定、快速地检测出蔗株是否感染RSD,且检测效率高,适合于田间大批量样品快速检测。The method of preparing DNA template was firstly improving,and then based on the basic program of PCR,used specific primers mapped among 16S-23S rDNA of Leifsonia xyli subsp.xyli,PCR reaction system was optimized by adjusting the concentration of each component in the reaction system,the annealing temperature and the cycle parameters etc.Using the optimized reaction system,specific fragments in the size of 438 bp were amplified from all the samples infected with RSD,whereas there was no specific fragment from the negative control and the non-infected samples;the specific fragments can be amplified even DNA template was diluted by 3 000 times;the detection results for 31 samples collected from different fields using the optimized method were consistent with the results of I-ELISA,the positive rates were 32.26% in the unoptimized method and 74.19% in the optimized method.The optimized method can be used for determining whether the plants were infected with RSD stably and rapidly,and the detection efficiency was very high;therefore,it is suitable for the short-term detection for large quantities of field samples.

关 键 词：甘蔗宿根矮化病菌 PCR检测 16S-23SrDNA 间接酶联免疫吸附法 

分 类 号：S435.661[农业科学—农业昆虫与害虫防治]