scholarly journals Taxonomic and Strain-Specific Identification of the Probiotic Strain Lactobacillus rhamnosus 35 within the Lactobacillus casei Group

2008 ◽  
Vol 74 (9) ◽  
pp. 2679-2689 ◽  
Author(s):  
Sophie Coudeyras ◽  
Hélène Marchandin ◽  
Céline Fajon ◽  
Christiane Forestier
Keyword(s):  
Gel Electrophoresis ◽  
Lactobacillus Casei ◽  
Group Analysis ◽  
Lactobacillus Rhamnosus ◽  
Probiotic Strain ◽  
Subtractive Hybridization ◽  
Specific Pcr ◽  
Gradient Gel Electrophoresis ◽  
Species Type ◽  
Temperature Gradient Gel Electrophoresis

ABSTRACT Lactobacilli are lactic acid bacteria that are widespread in the environment, including the human diet and gastrointestinal tract. Some Lactobacillus strains are regarded as probiotics because they exhibit beneficial health effects on their host. In this study, the long-used probiotic strain Lactobacillus rhamnosus 35 was characterized at a molecular level and compared with seven reference strains from the Lactobacillus casei group. Analysis of rrn operon sequences confirmed that L. rhamnosus 35 indeed belongs to the L. rhamnosus species, and both temporal temperature gradient gel electrophoresis and ribotyping showed that it is closer to the probiotic strain L. rhamnosus ATCC 53103 (also known as L. rhamnosus GG) than to the species type strain. In addition, L. casei ATCC 334 gathered in a coherent cluster with L. paracasei type strains, unlike L. casei ATCC 393, which was closer to L. zeae; this is evidence of the lack of relatedness between the two L. casei strains. Further characterization of the eight strains by pulsed-field gel electrophoresis repetitive DNA element-based PCR identified distinct patterns for each strain, whereas two isolates of L. rhamnosus 35 sampled 40 years apart could not be distinguished. By subtractive hybridization using the L. rhamnosus GG genome as a driver, we were able to isolate five L. rhamnosus 35-specific sequences, including two phage-related ones. The primer pairs designed to amplify these five regions allowed us to develop rapid and highly specific PCR-based identification methods for the probiotic strain L. rhamnosus 35.

scholarly journals Application of temperature gradient gel electrophoresis to the characterization of a nitrifying bioaugmentation product

2003 ◽  
Vol 43 (2) ◽  
pp. 277-286 ◽  
Author(s):  
Melissa A. Fouratt ◽  
Jeremy S. Rhodes ◽  
Charles M. Smithers ◽  
Nancy G. Love ◽  
Ann M. Stevens
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis

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 Denaturing gradient gel electrophoresis as a fingerprinting method for the analysis of soil microbial communities

2009 ◽  
Vol 55 (No. 10) ◽  
pp. 413-423 ◽  
Author(s):  
V. Valášková ◽  
P. Baldrian
Keyword(s):  
Microbial Communities ◽  
Gel Electrophoresis ◽  
Denaturing Gradient Gel Electrophoresis ◽  
Soil Microbial Communities ◽  
Soil Microbial ◽  
Genes Encoding ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis ◽  
Experimental Treatments ◽  
Multiple Samples

In soil microbial ecology, the effects of environmental factors and their gradients, temporal changes or the response to specific experimental treatments of microbial communities can only be effectively analyzed using methods that address the structural differences among whole communities. Fingerprinting methods are the most appropriate technique for this task when multiple samples must be analyzed. Among the methods currently used to compare microbial communities based on nucleic acid sequences, the techniques based on differences in the melting properties of double-stranded molecules, denaturing gradient gel electrophoresis (DGGE) or temperature gradient gel electrophoresis (TGGE), are the most widely used. Their main advantage is that they provide the possibility to further analyze whole sequences contained in fingerprints using molecular methods. In addition to the analysis of microbial communities based on DNA extracted from soils, DGGE/TGGE can also be used for the assessment of the active part of the community based on the analysis of RNA-derived sequences or for the analysis of sequences of functional genes encoding for proteins involved in important soil processes.

Temporal Temperature Gradient Gel Electrophoresis

2004 ◽  
pp. 1257-1261
Author(s):  
Lee‐Jun C. Wong ◽  
Özgül Alper ◽  
Hae‐Young Kwon ◽  
Duan‐Jun Tan ◽  
Tian‐Jian Chen
Keyword(s):  
Temperature Gradient ◽  
Gel Electrophoresis ◽  
Gradient Gel Electrophoresis ◽  
Temperature Gradient Gel Electrophoresis
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