Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing.
Improved detection of bifidobacteria with optimised 16S rRNA-gene based pyrosequencing.
复制标题
DOI：
10.1371/journal.pone.0032543
复制
发表时间：
2012
期刊：
影响因子：
3.7
通讯作者：
Kroll JS
中科院分区：
文献类型：
--
作者：
Sim K;Cox MJ;Wopereis H;Martin R;Knol J;Li MS;Cookson WO;Moffatt MF;Kroll JS
16S rRNA基因在所有细菌中是保守的，因此在细菌多样性的PCR调查中是常规靶向的。PCR引物设计的目的是从尽可能广泛的生物体中扩增尽可能多的不同16S rRNA基因序列，尽管没有合适的100%保守的基因区域，导致偏倚。在胃肠道中，双歧杆菌是一个关键属，但在16S rRNA多样性调查中往往代表性不足。我们已经设计了修改后的，“双歧杆菌优化的”通用引物，我们已经证明了检测的双歧杆菌序列存在于DNA混合物中的丰度为2%，测试的最低比例。优化并不影响婴儿粪便样本中其他微生物的检测。使用荧光原位杂交（FISH）的单独验证表明，粪便样品中检测到的双歧杆菌的比例与使用基于16S rRNA的焦磷酸测序获得的比例一致。对于未来针对粪便微生物群的研究，仔细选择引物将是关键，以确保有效检测生物多样性细菌。
The 16S rRNA gene is conserved across all bacteria and as such is routinely targeted in PCR surveys of bacterial diversity. PCR primer design aims to amplify as many different 16S rRNA gene sequences from as wide a range of organisms as possible, though there are no suitable 100% conserved regions of the gene, leading to bias. In the gastrointestinal tract, bifidobacteria are a key genus, but are often under-represented in 16S rRNA surveys of diversity. We have designed modified, ‘bifidobacteria-optimised’ universal primers, which we have demonstrated detection of bifidobacterial sequence present in DNA mixtures at 2% abundance, the lowest proportion tested. Optimisation did not compromise the detection of other organisms in infant faecal samples. Separate validation using fluorescence in situ hybridisation (FISH) shows that the proportions of bifidobacteria detected in faecal samples were in agreement with those obtained using 16S rRNA based pyrosequencing. For future studies looking at faecal microbiota, careful selection of primers will be key in order to ensure effective detection of bifidobacteria.
登录
查看更多内容
影响因子：
14.9
作者：
Ludwig, W;Strunk, O;Schleifer, KH
通讯作者：
Schleifer, KH
影响因子：
3.7
作者：
Hong PY;Lee BW;Aw M;Shek LP;Yap GC;Chua KY;Liu WT
通讯作者：
Liu WT
DOI：
10.1097/00005176-200501000-00007
发表时间：
2005-01-01
影响因子：
2.9
作者：
Knol, J;Scholtens, P;Wells, J
通讯作者：
Wells, J
DOI：
10.1073/pnas.0807920105
发表时间：
2008-11-18
影响因子：
11.1
作者：
Fierer, Noah;Hamady, Micah;Knight, Rob
通讯作者：
Knight, Rob
影响因子：
3.8
作者：
Boehm, G;Stahl, B;Moro, GE
通讯作者：
Moro, GE