Use of group-specific PCR primers for identification of chrysophytes by denaturing gradient gel electrophoresis

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.

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Design and Evaluation of PCR Primers for Analysis of Bacterial Populations in Wine by Denaturing Gradient Gel Electrophoresis

Applied and Environmental Microbiology ◽

10.1128/aem.69.11.6801-6807.2003 ◽

2003 ◽

Vol 69 (11) ◽

pp. 6801-6807 ◽

Cited By ~ 124

Author(s):

Isabel Lopez ◽

Fernanda Ruiz-Larrea ◽

Luca Cocolin ◽

Erica Orr ◽

Trevor Phister ◽

...

Keyword(s):

Lactic Acid ◽

16S Rdna ◽

Gel Electrophoresis ◽

Denaturing Gradient Gel Electrophoresis ◽

Bacterial Species ◽

Pcr Primers ◽

Fungal Species ◽

Bacterial Populations ◽

Gradient Gel Electrophoresis ◽

Primer Sets

ABSTRACT Denaturing gradient gel electrophoresis (DGGE) of PCR-amplified ribosomal DNA (rDNA) is routinely used to compare levels of diversity of microbial communities and to monitor population dynamics. While using PCR-DGGE to examine the bacteria in wine fermentations, we noted that several commonly used PCR primers for amplifying bacterial 16S rDNA also coamplified yeast, fungal, or plant DNA present in samples. Unfortunately, amplification of nonbacterial DNA can result in a masking of bacterial populations in DGGE profiles. To surmount this problem, we developed two new primer sets for specific amplification of bacterial 16S rDNA in wine fermentation samples without amplification of eukaryotic DNA. One primer set, termed WLAB1 and WLAB2, amplified lactic acid bacteria, while another, termed WBAC1 and WBAC2, amplified both lactic acid bacterial and acetic acid bacterial populations found in wine. Primer specificity and efficacy were examined with DNA isolated from numerous bacterial, yeast, and fungal species commonly found in wine and must samples. Importantly, both primer sets effectively distinguished bacterial species in wine containing mixtures of yeast and bacteria.

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

Applied and Environmental Microbiology ◽

10.1128/aem.67.2.504-513.2001 ◽

2001 ◽

Vol 67 (2) ◽

pp. 504-513 ◽

Cited By ~ 322

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.

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

Applied and Environmental Microbiology ◽

10.1128/aem.00151-06 ◽

2006 ◽

Vol 72 (8) ◽

pp. 5232-5238 ◽

Cited By ~ 37

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.

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