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RESEARCH PRODUCT
Characterization of Bacterial and Fungal Soil Communities by Automated Ribosomal Intergenic Spacer Analysis Fingerprints: Biological and Methodological Variability
Jean-christophe LataSylvie NazaretFranck PolyJean ThioulouseL. RanjardChristophe Mougelsubject
DNA BacterialRibosomal Intergenic Spacer analysisBiologyPolymerase Chain ReactionApplied Microbiology and Biotechnology03 medical and health sciencesIntergenic regionRNA Ribosomal 16SDNA Ribosomal SpacerMethodsDNA FungalComputingMilieux_MISCELLANEOUSEcosystemSoil Microbiology030304 developmental biology[SDV.EE]Life Sciences [q-bio]/Ecology environmentGenetics[ SDE.BE ] Environmental Sciences/Biodiversity and Ecology0303 health sciencesBacteriaEcology030306 microbiologyFungiReproducibility of ResultsGenes rRNASpacer DNABIOLOGIE MOLECULAIRERibosomal RNADNA FingerprintingDNA extraction[SDV.EE] Life Sciences [q-bio]/Ecology environmentRNA Ribosomal 23SDNA profilingRRNA Operon[SDE.BE]Environmental Sciences/Biodiversity and EcologySoil microbiologyFood ScienceBiotechnologydescription
ABSTRACT Automated rRNA intergenic spacer analysis (ARISA) was used to characterise bacterial (B-ARISA) and fungal (F-ARISA) communities from different soil types. The 16S-23S intergenic spacer region from the bacterial rRNA operon was amplified from total soil community DNA for B-ARISA. Similarly, the two internal transcribed spacers and the 5.8S rRNA gene (ITS1-5.8S-ITS2) from the fungal rRNA operon were amplified from total soil community DNA for F-ARISA. Universal fluorescence-labeled primers were used for the PCRs, and fragments of between 200 and 1,200 bp were resolved on denaturing polyacrylamide gels by use of an automated sequencer with laser detection. Methodological (DNA extraction and PCR amplification) and biological (inter- and intrasite) variations were evaluated by comparing the number and intensity of peaks (bands) between electrophoregrams (profiles) and by multivariate analysis. Our results showed that ARISA is a high-resolution, highly reproducible technique and is a robust method for discriminating between microbial communities. To evaluate the potential biases in community description provided by ARISA, we also examined databases on length distribution of ribosomal intergenic spacers among bacteria (L. Ranjard, E. Brothier, and S. Nazaret, Appl. Environ. Microbiol. 66:5334–5339, 2000) and fungi.
| year | journal | country | edition | language |
|---|---|---|---|---|
| 2001-10-01 | Applied and Environmental Microbiology |