土壤镰孢菌Real-Time QPCR定量方法的建立及应用  

Development and Application of a Real-Time Quantitative PCR Assay for Detection of Fusarium spp.in Soil

作　　者：魏巍[1,2,3] 许艳丽[1] 刘金波[1,3] 李春杰[1] 韩晓增[1] 李文滨[2] 李淑娴 

机构地区：[1]中国科学院东北地理与农业生态研究所,黑龙江哈尔滨150081 [2]大豆生物学教育部重点实验室,黑龙江哈尔滨150030 [3]中国科学院研究生院,北京100049 [4]Crop Genetics Research Unit(CGRU),United States Department of Agriculture-Agricultural Research Service(USDA-ARS),Stoneville 38776,USA

出　　处：《大豆科学》2010年第4期655-658,662,共5页Soybean Science

基　　金：中国科学院知识创新工程重大资助项目(KSCX1-YW-09-09);大豆生物学省部共建教育部重点实验室开放基金资助项目(SB08B01);国家“十一五”科技支撑计划资助项目(2006BAD21B01-15)

摘　　要：基于镰孢菌ITS序列,设计一对应用于实时荧光定量PCR(Real-Time QPCR)反应的镰孢菌属特异性引物TS和TR,并建立了相应的Real-Time QPCR体系。应用该反应体系,绝对定量了无肥(NF)、化肥(NP)以及化肥配施有机肥(NPM)等3种施肥措施下大豆田土壤镰孢菌DNA含量。结果表明:引物TS和TR对镰孢菌属真菌有较好的特异性;Real-Time QPCR反应的扩增曲线中各梯度浓度标准品的循环阈值(Ct值)间隔均匀,熔点曲线无杂峰,标准曲线的相关系数R2=0.993,斜率为-0.2927;在大豆生育时期的苗期,NF、NP及NPM措施下土壤镰孢菌总DNA每克干土中含量分别为18.33、44.61和140.83 pg,且NPM措施含量极显著高于NF及NP措施(P<0.01)。Base on the internal transcribed spacers（ITS） of Fusarium,a pair of specificity primers,TS and TR,were designed and synthesized for Real-time quantitative PCR（Real-Time QPCR） system of Fusarium spp.and then its optimal reaction system was established.Using this reaction system,the masses of genomic DNA of Fusarium spp.in soybean filed,which were extracted from three fertilization management,including no fertilizer（NF）,chemical N and P（NP） and chemical N,P and manure（NPM）,were quantified absolutely.TS and TR have possessed a good specificity to Fusarium spp..In the amplification curve of this Real-Time QPCR reaction,the spacings of Cycle Threshold（Ct） of standard substance under every concentration gradient were uniform.The melting curve showed a simple peak.The correlation coefficient（R2） and slope of the equation from the standard curve were respective 0.993 and-0.2927.At soybean seedling stage,the mass of genomic DNA of Fusarium spp.per gram of soil under NF,NP and NPM fertilization management were 18.33,44.61 and 140.83 pg,respectively.The mass in NPM measure was higher than NF and NP measure significantly（P0.01）.

关 键 词：镰孢菌 实时荧光定量PCR 绝对定量 

分 类 号：S432.44[农业科学—植物病理学]