Department of Food Science & Human Nutrition, University of Illinois, Urbana, IL 61801, United States.
J Microbiol Methods. 2012 Nov;91(2):262-8. doi: 10.1016/j.mimet.2012.08.005. Epub 2012 Aug 17.
Terminal restriction fragment length polymorphism (T-RFLP) analysis has been widely used for studying microbial communities. However, most T-RFLP assays use 16S rDNA as the target and are unable to accurately characterize a microbial subpopulation. In this study, we developed a novel T-RFLP protocol based on Lactobacillus hsp60 to rapidly characterize and compare lactobacilli composition. The theoretical terminal restriction fragment (TRF) profiles were calculated from 769 Lactobacillus hsp60 sequences from online databases. In silico digestion with restriction endonucleases AluI and TacI on hsp60 amplicons generated 83 distinct TRF patterns, of which, 70 were species specific. To validate the assay, five previously sequenced lactobacilli were cultured independently, mixed at known concentrations and subjected to analysis by T-RFLP. All five strains generated the predicted TRFs and a qualitative consistent relationship was revealed. We performed the T-RFLP protocol on fecal samples from mice fed 6 different diets (n=4). Principal component analysis and agglomerative hierarchical clustering revealed that the lactobacilli community was strongly connected to dietary supplementation. Our study demonstrates the potential for using Lactobacillus specific T-RFLP to characterize lactobacilli communities in complex samples.
末端限制性片段长度多态性（T-RFLP）分析已广泛用于研究微生物群落。然而，大多数 T-RFLP 检测方法使用 16S rDNA 作为靶标，无法准确地描述微生物亚群。在这项研究中，我们开发了一种基于乳杆菌 hsp60 的新型 T-RFLP 方案，用于快速表征和比较乳杆菌组成。从在线数据库中 769 个乳杆菌 hsp60 序列中计算出理论末端限制片段（TRF）图谱。在 hsp60 扩增子上用限制内切酶 AluI 和 TacI 进行计算机消化，产生了 83 种不同的 TRF 模式，其中 70 种是种特异性的。为了验证该检测方法，我们将 5 株先前测序的乳杆菌分别培养，以已知浓度混合，并进行 T-RFLP 分析。所有 5 株菌株均产生了预测的 TRF，并且显示出定性一致的关系。我们对饲喂 6 种不同饮食（n=4）的小鼠粪便样本进行了 T-RFLP 方案。主成分分析和凝聚层次聚类显示，乳杆菌群落与饮食补充密切相关。我们的研究表明，使用乳杆菌特异性 T-RFLP 来表征复杂样本中的乳杆菌群落具有潜力。
