Tambong J T, de Cock A W A M, Tinker N A, Lévesque C A
Agriculture and Agri-Food Canada, Environmental Health Program (Biodiversity), 960 Carling Avenue, Ottawa, Ontario K1A 0C6, Canada.
Appl Environ Microbiol. 2006 Apr;72(4):2691-706. doi: 10.1128/AEM.72.4.2691-2706.2006.
A DNA array containing 172 oligonucleotides complementary to specific diagnostic regions of internal transcribed spacers (ITS) of more than 100 species was developed for identification and detection of Pythium species. All of the species studied, with the exception of Pythium ostracodes, exhibited a positive hybridization reaction with at least one corresponding species-specific oligonucleotide. Hybridization patterns were distinct for each species. The array hybridization patterns included cluster-specific oligonucleotides that facilitated the recognition of species, including new ones, belonging to groups such as those producing filamentous or globose sporangia. BLAST analyses against 500 publicly available Pythium sequences in GenBank confirmed that species-specific oligonucleotides were unique to all of the available strains of each species, of which there were numerous economically important ones. GenBank entries of newly described species that are not putative synonyms showed no homology to sequences of the spotted species-specific oligonucleotides, but most new species did match some of the cluster-specific oligonucleotides. Further verification of the specificity of the DNA array was done with 50 additional Pythium isolates obtained by soil dilution plating. The hybridization patterns obtained were consistent with the identification of these isolates based on morphology and ITS sequence analyses. In another blind test, total DNA of the same soil samples was amplified and hybridized on the array, and the results were compared to those of 130 Pythium isolates obtained by soil dilution plating and root baiting. The 13 species detected by the DNA array corresponded to the isolates obtained by a combination of soil dilution plating and baiting, except for one new species that was not represented on the array. We conclude that the reported DNA array is a reliable tool for identification and detection of the majority of Pythium species in environmental samples. Simultaneous detection and identification of multiple species of soilborne pathogens such as Pythium species could be a major step forward for epidemiological and ecological studies.
开发了一种DNA芯片，其包含172个与100多种内部转录间隔区（ITS）的特定诊断区域互补的寡核苷酸，用于腐霉菌种的鉴定和检测。除了壳状腐霉外，所有研究的物种都与至少一种相应的物种特异性寡核苷酸表现出阳性杂交反应。每个物种的杂交模式都不同。阵列杂交模式包括簇特异性寡核苷酸，有助于识别属于产生丝状或球状孢子囊等类群的物种，包括新物种。对GenBank中500个公开可用的腐霉序列进行的BLAST分析证实，物种特异性寡核苷酸对于每个物种的所有可用菌株都是独特的，其中有许多具有重要经济意义的菌株。新描述的非假定同义词物种的GenBank条目与斑点物种特异性寡核苷酸的序列没有同源性，但大多数新物种确实与一些簇特异性寡核苷酸匹配。通过土壤稀释平板法获得的另外50株腐霉分离株对DNA芯片的特异性进行了进一步验证。获得的杂交模式与基于形态学和ITS序列分析对这些分离株的鉴定结果一致。在另一次盲测中，对相同土壤样品的总DNA进行扩增并在芯片上杂交，并将结果与通过土壤稀释平板法和根诱饵法获得的130株腐霉分离株的结果进行比较。DNA芯片检测到的13个物种与通过土壤稀释平板法和诱饵法组合获得的分离株相对应，除了芯片上未显示的一个新物种。我们得出结论，所报道的DNA芯片是用于鉴定和检测环境样品中大多数腐霉菌种的可靠工具。同时检测和鉴定多种土壤传播病原体，如腐霉菌种，可能是流行病学和生态学研究向前迈出的重要一步。
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