利用实时荧光定量PCR技术检测油菜菌核病菌  

Detection of Sclerotinia sclerotiorum by a quantitative real-time PCR

作　　者：陈长军[1] 李俊[1] 赵伟[1] 王建新[1] 周明国[1] 

机构地区：[1]南京农业大学植物保护学院植物病理学系,南京210095

出　　处：《植物病理学报》2011年第5期516-525,共10页Acta Phytopathologica Sinica

基　　金：国家"973"计划(2006CB101900);农业部农业行业专项(201103016);国家"863"计划(2006AA10A2112008AA102414);江苏省农业科技攻关项目(BE2006304)

摘　　要：采用高通量实时荧光定量PCR技术建立检测和监测油菜菌核病菌群体数量的方法。利用油菜菌核病菌(Sclerotiniasclerotiorum)的β-微管蛋白基因内含子序列的特异性,设计引物对SclSF(5'-CTCAAATCTCCGAAAGTT-3')/SclAF(5'-TGCAGACGGGTAATATG-3'),建立和优化了SYBR GreenⅠ实时荧光定量PCR检测体系。结果表明,该引物对能够从8种所测试的十字花科植物常见病原真菌中特异性扩增出油菜菌核病菌;所建立的实时定量PCR技术可应用于油菜病叶和病茎中菌核病菌的早期检测及菌核病的预测预报。A quantitative real-time PCR was used in high-throughout detection of Sclerotinia sclerotiorum populations.A pair of primers SclSF（5′-CTCAAATCTCCGAAAGTT-3′）/SclAF（5′-TGCAGACGGGTAATATG-3′） was developed and synthesized according to the specificity of the nucleotide sequences of introns of beta-tubulin gene from Sclerotinia sclerotiorum.A technique of quantitative realtime PCR by SYBR Green Ⅰwas developed and optimized to detect S.sclerotiorum.The results indicated that S.sclerotiorum could be differentiated by conventional PCR assay with the developed primer set SclSF/SclAF from other eight crucifer pathogens tested,including Alternaria oleracea,Cercosporella albo-maculans,Phoma lingam,Albugo bliti,Botrytis cinerea,Colletotrichum gloeosporioides,Peronospora parasitica and Erysiphe cruciferarum.The real-time PCR technique appears suitable for early detection and the epidemiological studies of S.sclerotiorum from diseased stems and leaves of oilseed rape.

关 键 词：油菜菌核病菌 实时定量PCR SYBR GreenⅠ 

分 类 号：S436.3[农业科学—农业昆虫与害虫防治]