McBurney W, Mangold M, Munro K, Schultz M, Rath H C, Tannock G W
Department of Microbiology and Immunology, University of Otago, Dunedin, New Zealand.
Lett Appl Microbiol. 2006 Feb;42(2):165-71. doi: 10.1111/j.1472-765X.2005.01811.x.
To determine the phylogenetic composition of the colonic microbiota of transgenic (TG) HLA-B27 rats using 16S ribosomal RNA (rRNA) gene sequences obtained from denaturing gradient gel electrophoresis (DGGE) gels and sequences from a 16S rRNA gene library.
Colonic microbiota of TG and nontransgenic (NT) rats harboured by 10-week-old and 6-month-old animals was screened using PCR/DGGE. Six months old TG rats had marked inflammation of the colon compared with 10-week-old TG and NT rats. The DGGE profiles of rats with inflamed colon were similar from rat to rat (Dice's Similarity Coefficient proximal colon 73%, distal colon 83%) whereas profiles from animals without inflammation were dissimilar (52-64%). Identifications of bacterial origins of 16S rRNA gene sequences obtained from DGGE gels (200 bp) and from 16S rRNA clones (450 bp) of the colonic microbiota of diseased rats gave sequences most closely phylogenetically affiliated with uncultured or unknown bacteria.
PCR/DGGE was shown to be an effective method to compare the colonic microbiota composition of TG and NT rats relative to the progression of inflammatory disease. Sequencing of 16S rRNA gene fragments from DGGE gels or 16S rRNA gene clones from a random library showed that uncultured or unknown bacteria were most commonly detected by both methods. It can be concluded that it would be better in future studies to search for the antigens produced by the gut microbiota against which the dysfunctional immune system reacts rather than seek phylogenetic associations.
PCR/DGGE can be used as a rapid initial screening method to compare the composition of bacterial communities of initially unknown composition that are associated with the development of intestinal disease.
利用从变性梯度凝胶电泳（DGGE）凝胶获得的16S核糖体RNA（rRNA）基因序列以及16S rRNA基因文库中的序列，确定转基因（TG）HLA - B27大鼠结肠微生物群的系统发育组成。
使用PCR/DGGE对10周龄和6月龄动物体内的TG和非转基因（NT）大鼠的结肠微生物群进行筛选。与10周龄的TG和NT大鼠相比，6月龄的TG大鼠结肠有明显炎症。结肠发炎大鼠的DGGE图谱彼此相似（近端结肠的戴斯相似系数为73%，远端结肠为83%），而无炎症动物的图谱则不同（52 - 64%）。对患病大鼠结肠微生物群从DGGE凝胶（200 bp）和16S rRNA克隆（450 bp）获得的16S rRNA基因序列进行细菌溯源鉴定，结果显示其序列在系统发育上与未培养或未知细菌关系最为密切。
PCR/DGGE被证明是一种有效的方法，可用于比较TG和NT大鼠结肠微生物群组成与炎症性疾病进展的关系。对DGGE凝胶中的16S rRNA基因片段或随机文库中的16S rRNA基因克隆进行测序表明，两种方法最常检测到的是未培养或未知细菌。可以得出结论，在未来的研究中，更好的做法是寻找肠道微生物群产生的、功能失调的免疫系统会对其产生反应的抗原，而不是寻找系统发育关联。
PCR/DGGE可作为一种快速的初始筛选方法，用于比较与肠道疾病发展相关的、初始组成未知的细菌群落的组成。
