scholarly journals Bifidobacterial Diversity in Human Feces Detected by Genus-Specific PCR and Denaturing Gradient Gel Electrophoresis

2001 ◽  
Vol 67 (2) ◽  
pp. 504-513 ◽  
Author(s):  
Reetta M. Satokari ◽  
Elaine E. Vaughan ◽  
Antoon D. L. Akkermans ◽  
Maria Saarela ◽  
Willem M. de Vos
Keyword(s):  
16S Rdna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Methodological Approach ◽  
Common Species ◽  
Bifidobacterium Adolescentis ◽  
Specific Pcr ◽  
Rdna Sequences ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis

ABSTRACT We describe the development and validation of a method for the qualitative analysis of complex bifidobacterial communities based on PCR and denaturing gradient gel electrophoresis (DGGE).Bifidobacterium genus-specific primers were used to amplify an approximately 520-bp fragment from the 16S ribosomal DNA (rDNA), and the fragments were separated in a sequence-specific manner in DGGE. PCR products of the same length from different bifidobacterial species showed good separation upon DGGE. DGGE of fecal 16S rDNA amplicons from five adult individuals showed host-specific populations of bifidobacteria that were stable over a period of 4 weeks. Sequencing of fecal amplicons resulted in Bifidobacterium-like sequences, confirming that the profiles indeed represent the bifidobacterial population of feces. Bifidobacterium adolescentis was found to be the most common species in feces of the human adult subjects in this study. The methodological approach revealed intragenomic 16S rDNA heterogeneity in the type strain of B. adolescentis, E-981074. The strain was found to harbor five copies of 16S rDNA, two of which were sequenced. The two 16S rDNA sequences of B. adolescentis E-981074T exhibited microheterogeneity differing in eight positions over almost the total length of the gene.

scholarly journals Molecular Profiling of the Clostridium leptum Subgroup in Human Fecal Microflora by PCR-Denaturing Gradient Gel Electrophoresis and Clone Library Analysis

2006 ◽  
Vol 72 (8) ◽  
pp. 5232-5238 ◽  
Author(s):  
Jian Shen ◽  
Baorang Zhang ◽  
Guifang Wei ◽  
Xiaoyan Pang ◽  
Hua Wei ◽  
...  
Keyword(s):  
Clone Library ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Clone Library Analysis ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Universal Bacterial Primer ◽  
Group Specific Primer

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, ∼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.

scholarly journals Evaluations of Different Hypervariable Regions of Archaeal 16S rRNA Genes in Profiling of Methanogens by Archaea-Specific PCR and Denaturing Gradient Gel Electrophoresis

2007 ◽  
Vol 74 (3) ◽  
pp. 889-893 ◽  
Author(s):  
Zhongtang Yu ◽  
Rubén García-González ◽  
Floyd L. Schanbacher ◽  
Mark Morrison
Keyword(s):  
16S Rrna ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
16S Rrna Genes ◽  
Rrna Genes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
V Region

ABSTRACT Different hypervariable (V) regions of the archaeal 16S rRNA gene (rrs) were compared systematically to establish a preferred V region(s) for use in Archaea-specific PCR-denaturing gradient gel electrophoresis (DGGE). The PCR products of the V3 region produced the most informative DGGE profiles and permitted identification of common methanogens from rumen samples from sheep. This study also showed that different methanogens might be detected when different V regions are targeted by PCR-DGGE. Dietary fat appeared to transiently stimulate Methanosphaera stadtmanae but inhibit Methanobrevibacter sp. strain AbM4 in rumen samples.

scholarly journals Development and Validation of a Nested-PCR-Denaturing Gradient Gel Electrophoresis Method for Taxonomic Characterization of Bifidobacterial Communities

2003 ◽  
Vol 69 (11) ◽  
pp. 6380-6385 ◽  
Author(s):  
R. Temmerman ◽  
L. Masco ◽  
T. Vanhoutte ◽  
G. Huys ◽  
J. Swings
Keyword(s):  
Nested Pcr ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Temporal Changes ◽  
Rrna Gene ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Specific ◽  
Taxonomic Characterization

ABSTRACT The taxonomic characterization of a bacterial community is difficult to combine with the monitoring of its temporal changes. None of the currently available identification techniques are able to visualize a “complete” community, whereas techniques designed for analyzing bacterial ecosystems generally display limited or labor-intensive identification potential. This paper describes the optimization and validation of a nested-PCR-denaturing gradient gel electrophoresis (DGGE) approach for the species-specific analysis of bifidobacterial communities from any ecosystem. The method comprises a Bifidobacterium-specific PCR step, followed by purification of the amplicons that serve as template DNA in a second PCR step that amplifies the V3 and V6-V8 regions of the 16S rRNA gene. A mix of both amplicons is analyzed on a DGGE gel, after which the band positions are compared with a previously constructed database of reference strains. The method was validated through the analysis of four artificial mixtures, mimicking the possible bifidobacterial microbiota of the human and chicken intestine, a rumen, and the environment, and of two fecal samples. Except for the species Bifidobacterium coryneforme and B. indicum, all currently known bifidobacteria originating from various ecosystems can be identified in a highly reproducible manner. Because no further cloning and sequencing of the DGGE bands is necessary, this nested-PCR-DGGE technique can be completed within a 24-h span, allowing the species-specific monitoring of temporal changes in the bifidobacterial community.

scholarly journals Molecular Analysis of Ammonia-Oxidizing Bacteria of the β Subdivision of the Class Proteobacteria in Compost and Composted Materials

1999 ◽  
Vol 65 (2) ◽  
pp. 396-403 ◽  
Author(s):  
George A. Kowalchuk ◽  
Zinaida S. Naoumenko ◽  
Piet J. L. Derikx ◽  
Andreas Felske ◽  
John R. Stephen ◽  
...  
Keyword(s):  
Denaturing Gradient Gel Electrophoresis ◽  
Ammonia Oxidizer ◽  
Ammonia Oxidizing Bacteria ◽  
Nitrogen Transformations ◽  
Rt Pcr ◽  
Gene Encoding ◽  
Specific Pcr ◽  
Pcr Products ◽  
Gradient Gel Electrophoresis ◽  
Almost All

ABSTRACT Although the practice of composting animal wastes for use as biofertilizers has increased in recent years, little is known about the microorganisms responsible for the nitrogen transformations which occur in compost and during the composting process. Ammonia is the principle available nitrogenous compound in composting material, and the conversion of this compound to nitrite in the environment by chemolithotrophic ammonia-oxidizing bacteria is an essential step in nitrogen cycling. Therefore, the distribution of ammonia-oxidizing members of the β subdivision of the class Proteobacteriain a variety of composting materials was assessed by amplifying 16S ribosomal DNA (rDNA) and 16S rRNA by PCR and reverse transcriptase PCR (RT-PCR), respectively. The PCR and RT-PCR products were separated by denaturing gradient gel electrophoresis (DGGE) and were identified by hybridization with a hierarchical set of oligonucleotide probes designed to detect ammonia oxidizer-like sequence clusters in the genera Nitrosospira and Nitrosomonas. Ammonia oxidizer-like 16S rDNA was detected in almost all of the materials tested, including industrial and experimental composts, manure, and commercial biofertilizers. A comparison of the DGGE and hybridization results after specific PCR and RT-PCR suggested that not all of the different ammonia oxidizer groups detected in compost are equally active. amoA, the gene encoding the active-site-containing subunit of ammonia monooxygenase, was also targeted by PCR, and template concentrations were estimated by competitive PCR. Detection of ammonia-oxidizing bacteria in the composts tested suggested that such materials may not be biologically inert with respect to nitrification and that the fate of nitrogen during composting and compost storage may be affected by the presence of these organisms.

scholarly journals Distribution and Diversity of Archaea Corresponding to the Limnological Cycle of a Hypersaline Stratified Lake (Solar Lake, Sinai, Egypt)

2000 ◽  
Vol 66 (8) ◽  
pp. 3269-3276 ◽  
Author(s):  
Eddie Cytryn ◽  
Dror Minz ◽  
Ronald S. Oremland ◽  
Yehuda Cohen
Keyword(s):  
Phylogenetic Analysis ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Archaeal Community ◽  
Seasonal Distribution ◽  
Stratified Lake ◽  
Rdna Sequences ◽  
16S Rdna Sequences ◽  
Gradient Gel Electrophoresis ◽  
Solar Lake

ABSTRACT The vertical and seasonal distribution and diversity of archaeal sequences was investigated in a hypersaline, stratified, monomictic lake, Solar Lake, Sinai, Egypt, during the limnological development of stratification and mixing. Archaeal sequences were studied via phylogenetic analysis of 16S rDNA sequences as well as denaturing gradient gel electrophoresis analysis. The 165 clones studied were grouped into four phylogenetically different clusters. Most of the clones isolated from both the aerobic epilimnion and the sulfide-rich hypolimnion were defined as cluster I, belonging to theHalobacteriaceae family. The three additional clusters were all isolated from the anaerobic hypolimnion. Cluster II is phylogenetically located between the generaMethanobacterium and Methanococcus. Clusters III and IV relate to two previously documented groups of uncultured euryarchaeota, remotely related to the genusThermoplasma. No crenarchaeota were found in the water column of the Solar Lake. The archaeal community in the Solar Lake under both stratified and mixed conditions was dominated by halobacteria in salinities higher than 10%. During stratification, additional clusters, some of which may possibly relate to uncultured halophilic methanogens, were found in the sulfide- and methane-rich hypolimnion.

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