title: Molecular profiling of the Clostridium leptum subgroup in human fecal microflora by PCR-denaturing gradient gel electrophoresis and clone library analysis.
abstract: A group-specific PCR-based denaturing gradient gel electrophoresis (DGGE) method was developed and combined with group-specific clone library analysis to investigate the diversity of the Clostridium leptum subgroup in human feces. PCR products (length, 239 bp) were amplified using C. leptum cluster-specific primers and were well separated by DGGE. The DGGE patterns of fecal amplicons from 11 human individuals revealed host-specific profiles; the patterns for fecal samples collected from a child for 3 years demonstrated the structural succession of the population in the first 2 years and its stability in the third year. A clone library was constructed with 100 clones consisting of 1,143-bp inserts of 16S rRNA gene fragments that were amplified from one adult fecal DNA with one forward universal bacterial primer and one reverse group-specific primer. Eighty-six of the clones produced the 239-bp C. leptum cluster-specific amplicons, and the remaining 14 clones did not produce these amplicons but still phylogenetically belong to the subgroup. Sixty-four percent of the clones were related to Faecalibacterium prausnitzii (similarity, 97 to 99%), 6% were related to Subdoligranulum variabile (similarity, approximately 99%), 2% were related to butyrate-producing bacterium A2-207 (similarity, 99%), and 28% were not identified at the species level. The identities of most bands in the DGGE profiles for the same adult were determined by comigration analysis with the 86 clones that harbored the 239-bp group-specific fragments. Our results suggest that DGGE combined with clone library analysis is an effective technique for monitoring and analyzing the composition of this important population in the human gut flora.
authors.affiliation: Laboratory of Molecular Microbial Ecology and Ecogenomics, College of Life Science and Biotechnology, Shanghai Jiaotong University, Shanghai 200240, China.
authors.forename: Jian
authors.initials: J
authors.lastname: Shen
authors.forename: Baorang
authors.initials: B
authors.lastname: Zhang
authors.forename: Guifang
authors.initials: G
authors.lastname: Wei
authors.forename: Xiaoyan
authors.initials: X
authors.lastname: Pang
authors.forename: Hua
authors.initials: H
authors.lastname: Wei
authors.forename: Min
authors.initials: M
authors.lastname: Li
authors.forename: Yan
authors.initials: Y
authors.lastname: Zhang
authors.forename: Wei
authors.initials: W
authors.lastname: Jia
authors.forename: Liping
authors.initials: L
authors.lastname: Zhao
mesh_terms: D000328:Adult; D002648:Child; D002675:Child, Preschool; D003001:Cloning, Molecular / Q000379:methods*; D003013:Clostridium / Q000145:classification* / Q000235:genetics; D004591:Electrophoresis, Polyacrylamide Gel / Q000379:methods*; D005243:Feces / Q000382:microbiology*; D005260:Female; D015723:Gene Library; D014644:Genetic Variation; D006801:Humans; D008297:Male; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA