三重PCR检测草莓灰霉病菌、炭疽病菌和黄萎病菌  

Triplex PCR Detection of Botrytis cinerea,Colletotrichum gloeosporioides and Verticillium dahliae in Infected Strawberry Plant Tissues

作　　者：王楠[1] 王剑[1] 尹丹韩[1] 高观朋[1] 王伟[1] 

机构地区：[1]华东理工大学/生物反应器工程国家重点实验室,上海200237

出　　处：《中国农业科学》2010年第21期4392-4400,共9页Scientia Agricultura Sinica

基　　金：国家自然科学基金项目(30871664);上海市重点攻关项目(08391911400);国家重点实验室专项经费(2060204)

摘　　要：【目的】探索和优化检测条件,建立同时检测草莓灰霉病菌Botrytiscinerea,炭疽病菌Colletotrichum gloeosporioides和黄萎病菌Verticillium dahliae的三重PCR检测体系,为3种病害的早期快速诊断和鉴定提供技术和方法。【方法】选择可以组合的3种病原菌特异引物,研究多重PCR的影响因素,优化PCR退火温度,采用正交试验方法,以3个引物组、TaqDNA聚合酶、dNTP和Mg2+六因素三水平优化多重PCR体系。【结果】建立并验证适合上述草莓主要病原菌的三重PCR最佳检测体系,可分别扩增出729、539和450bp的特异条带,最适退火温度为50℃,25μL的反应体系中含有0.32μmol·L-1C729+/-、0.032μmol·L-1DB19/DB22、0.32μmol·L-1CgInt/ITS4、1.5UTaq聚合酶、0.15mmol·L-1dNTP、1.6mmol·L-1MgCl2。【结论】利用上述引物组合和反应体系进行三重PCR检测,能够快速从田间发病植株和土壤中将草莓灰霉病菌、草莓炭疽病菌和草莓黄萎病菌检测出来,灵敏度可以达到10pg菌丝DNA。【Objective】 Reaction components and parameters of triplex PCR system were optimized to establish the method for the detection of Botrytis cinerea, Colletotrichum gloeosporioides and Verticillium dahliae simultaneously at early stage. 【Method】Three sets of specific primers were selected and the annealing temperature of PCR reaction was optimized. The three levels of six factors （three primers, Taq DNA polymerase, dNTP and Mg2＋） in multiplex PCR system were evaluated using orthogonal design method. 【Result】 The triplex PCR system for simultaneous detection of these three pathogens in strawberry was successfully established and verified. The volume of reaction system which could amplify three specific bands of 729 bp, 539 bp and 450 bp was 25 μL and the annealing temperature was 50℃. The reaction mix contained 0.32 μmol·L-1 of primer C729＋/-, 0.032 μmol·L-1 of primer DB19/DB22, 0.32 μmol·L-1 of primer CgInt/ITS4, 1.5 U Taq DNA polymerase, 0.15 mmol·L-1 dNTP and 1.6 mmol·L-1 MgCl2. 【Conclusion】 The triplex PCR system can detect B. cinerea, C. gloeosporioides and V. dahliae in infected plant tissues and soil with the sensitivity of 10 pg DNA.

关 键 词：草莓 灰霉病菌 炭疽病菌 黄萎病菌 分子检测 三重PCR 

分 类 号：S436.68[农业科学—农业昆虫与害虫防治]