向日葵黑茎病双重PCR检测实用化研究  

Application Studies on Duplex PCR Detection of Sunflower Black Stem Pathogen

作　　者：米瑶[1] 李培江[1] 张伟宏[1] 王香[1] 廖芳[2] 李关荣[1] 

机构地区：[1]西南大学农学与生物科技学院,重庆400716 [2]天津出入境检验检疫局,天津300461

出　　处：《西南农业学报》2015年第5期2103-2106,共4页Southwest China Journal of Agricultural Sciences

基　　金：科技部"十二五"科技支撑项目(2012BAK11B02);中华人民共和国质检总局项目(2012IK286;2013IK290)

摘　　要：本研究采用从新疆田间分离经ITS序列扩增、测序鉴定了的黑茎病菌株BXC1为供试菌,对其双重PCR分子检测、灵敏度试验以及模拟带菌组织的分子检测,再将此菌株采用不同接种方式人工接种2种向日葵品种幼苗,待其出现典型症状时,对其进行双重PCR实际检测。结果表明,无论是黑茎病菌基因组DNA、还是其与向日葵基因组的混合DNA都能扩增出真菌的ITS区特异带(580 bp)和黑茎病菌Actin基因的特异带(255 bp),且混合DNA还能扩增出向日葵的ITS特异带(约740 bp),说明向日葵黑茎病菌的此种双重PCR分子检测方法具有很好的特异性;梯级稀释真菌DNA或向日葵基因组DNA或其混合DNA模板的双重PCR检测表明,在20μl PCR反应体系中,黑茎病菌DNA的检测灵敏度均为0.05 ng/μl;接种了BXC1的向日葵,其带菌组织DNA均能检测出向日葵与黑茎病的ITS序列以及黑茎病Actin基因特异条带。这些结果表明,此向日葵黑茎病菌的双重PCR检测体系特异、可靠、便捷,可对带菌向日葵组织进行直接快速分子检测。In this paper,the strain BXC1 of Leptosphaeria lindquistii separated from the rural field in Xinjiang and preliminarily identified by its ITS amplification and sequencing was used for duplex PCR detection,sensitivity test and mock detection of mixed sunflower DNA and the fungal DNA,and then the duplex PCR detections on the seedlings of two sunflower varieties challenged by BXC1 with different inoculation methods were conducted when its typical symptom appeared. The results showed that both the genomic DNA of BXC1 and its mixture with sunflower genomic DNA could produce the fungal ITS and the Actin gene specific bands（ 580 and 255 bp,respectively） by the duplex PCR.In addition,the mixture DNA could also produce the sunflower ITS specific band（ 740 bp）,indicating the excellent specificity of this established duplex PCR detection of Leptosphaeria lindquistii; Using the gradually diluted BXC1 DNA or sunflower DNA or the mixture as templates to perform the duplex PCR,a sensitivity of 0. 05 ng fungal DNA / μl in 20 μl PCR reaction was detected; The stem portions of the inoculated seedlings of the two sunflower varieties all produced the fungal ITS and the Actin gene specific bands by the established duplex PCR detection. These results indicated that the duplex PCR detection of Leptosphaeria lindquistii was specific,reliable and convenient and could be used directly for the quick detection of Leptosphaeria lindquistii pathogen in the infected sunflower tissues.

关 键 词：向日葵黑茎病 BXC1菌株 双重PCR检测 灵敏度试验 带菌组织检测 

分 类 号：S435.655[农业科学—农业昆虫与害虫防治]