Miyagi University of Education, Aramaki-aza-Aoba, Aoba-ku, Sendai 980-0845, Japan.
Microbes Environ. 2012;27(2):136-41. doi: 10.1264/jsme2.me11287.
Ciliates play important roles as prey and predators in ecosystems. Changes in the ciliate community can affect the composition and population of microfauna and microflora in ecosystems. To investigate the structure of ciliate communities, we developed a nested PCR-DGGE method, which combines a universal eukaryotic-specific primer set in the first PCR step with a ciliate-specific primer set in the second PCR step, to amplify 18S rRNA genes from ciliates. The 300 bp DGGE fragments generated more bands on the gel than the 600 bp DGGE fragments. Prior to bead beating, DNA extraction of ciliates from soil samples was optimized with a combination of freeze-thaw cycles and ultrasonication. We applied this nested PCR-DGGE method to agricultural soils amended with 0, 120, 300, and 600 t ha⁻¹ year⁻¹ of livestock slurry. The results from the DGGE profiles and principal component analysis (PCA) revealed that the supplement of slurry to soils influenced the ciliate communities. From phylogenetic analysis, 108 DGGE bands were assigned to six classes, which included Spirotrichea and Colpodea, of the subphylum Intramacronucleata, and one class of the subphylum Postciliodesmatophora. These results indicated that a wide variety of taxonomic groups were detected by DGGE profiling. Thus, the nested PCR-DGGE method described here could clearly differentiate between ciliate communities within soil samples and allowed for the phylogenetic identification of these ciliates at the class level.
纤毛虫在生态系统中作为猎物和捕食者起着重要作用。纤毛虫群落的变化会影响生态系统中微型动物和微生物的组成和数量。为了研究纤毛虫群落的结构，我们开发了一种嵌套 PCR-DGGE 方法，该方法将第一 PCR 步骤中的通用真核生物特异性引物与第二 PCR 步骤中的纤毛虫特异性引物结合，扩增纤毛虫的 18S rRNA 基因。与 600 bp 的 DGGE 片段相比，300 bp 的 DGGE 片段在凝胶上产生了更多的条带。在进行珠子研磨之前，我们通过冻融循环和超声处理相结合的方式优化了从土壤样品中提取纤毛虫的 DNA。我们将这种嵌套 PCR-DGGE 方法应用于添加了 0、120、300 和 600 t ha⁻¹ year⁻¹ 牲畜粪肥的农业土壤中。DGGE 图谱和主成分分析（PCA）的结果表明，粪肥的添加会影响纤毛虫群落。从系统发育分析来看，108 个 DGGE 条带被分配到六个类群，包括亚门内大核亚门的旋口纲和吸管纲，以及后纤毛亚门的一个类群。这些结果表明，DGGE 图谱分析检测到了广泛的分类群。因此，这里描述的嵌套 PCR-DGGE 方法可以清楚地区分土壤样品中的纤毛虫群落，并允许在类水平上对这些纤毛虫进行系统发育鉴定。
