Grönberg Henrietta, Paulin Lars, Sen Robin
Division of General Microbiology, Department of Biosciences, Viikki Biocenter, P.O. Box 56, FIN-00014 University of Helsinki, Finland.
Mycol Res. 2003 Apr;107(Pt 4):428-38. doi: 10.1017/s0953756203007500.
Development of specific DNA probes targeting rDNA internal transcribed spacer (ITS-1 or -2) sequences is described for the detection of strains representing uninucleate and binucleate Rhizoctonia spp. and Suillus bovinus. Discriminatory taxon/species-specific target sequences were identified following full length ITS sequence alignment of the test fungal sequences and those of other root associated pathogenic or mycorrhizal fungi. Both long (124-151 bp) and shorter (20-25 bp) probes were generated for assessment in Southern dot blot and liquid hybridization ITS capture-fragment length polymorphism assays. Fungal genomic DNA was presented as the target in dot blot protocols using the longer DIG (digoxigenin) labelled probes whilst the shorter 3' biotin-labelled oligonucleotide probes were hybridized to PCR amplified full length ITS (ITS1-5.8S-ITS2) in both dot blot and liquid hybridization assays. The optimal hybridization temperatures for dot blot detection also produced maximal target specific signals in the liquid hybridization protocol. The latter protocol was found to be more discriminatory as target fungi were detected on the basis of combined probe hybridization-ITS capture and 5' Cy-5 labelled ITS length polymorphism analysis (+/- 5 bp) following denaturing sequencing gel electrophoresis in a ALFexpress DNA sequencer.
本文描述了针对核糖体DNA内转录间隔区（ITS-1或-2）序列开发的特异性DNA探针，用于检测单核和双核丝核菌属以及牛肝菌的菌株。在对测试真菌序列以及其他与根相关的致病或菌根真菌的全长ITS序列进行比对后，确定了具有鉴别性的分类群/物种特异性靶序列。制备了长（124 - 151 bp）短（20 - 25 bp）两种探针，用于Southern斑点杂交和液相杂交ITS捕获 - 片段长度多态性分析。在斑点杂交实验中，使用较长的地高辛（DIG）标记探针将真菌基因组DNA作为靶标，而在斑点杂交和液相杂交实验中，较短的3'生物素标记寡核苷酸探针则与PCR扩增的全长ITS（ITS1 - 5.8S - ITS2）杂交。斑点杂交检测的最佳杂交温度在液相杂交实验中也产生了最大的靶标特异性信号。发现后一种实验方法更具鉴别性，因为在ALFexpress DNA测序仪中进行变性测序凝胶电泳后，基于探针杂交 - ITS捕获和5' Cy - 5标记的ITS长度多态性分析（±5 bp）来检测靶标真菌。
