TY - JOUR
T1 - Development of a Lactobacillus specific T-RFLP method to determine lactobacilli diversity in complex samples
AU - Chen, Long
AU - Teasdale, Matt T.
AU - Kaczmarczyk, Melissa M.
AU - Freund, Gregory G
AU - Miller, Michael J.
N1 - Funding Information:
This project was supported by the USDA National Institute of Food and Agriculture (Hatch project #ILLU-698-339 to MJM) and the National Institutes of Health ( DK064862 , NS058525 and AA019357 to GGF).
PY - 2012/11
Y1 - 2012/11
N2 - 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.
AB - 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.
KW - Community research
KW - Lactobacillus hsp60
KW - Terminal restriction fragment length polymorphism
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U2 - 10.1016/j.mimet.2012.08.005
DO - 10.1016/j.mimet.2012.08.005
M3 - Article
C2 - 22981747
AN - SCOPUS:84867831126
SN - 0167-7012
VL - 91
SP - 262
EP - 268
JO - Journal of Microbiological Methods
JF - Journal of Microbiological Methods
IS - 2
ER -