title: Use of PCR-DHPLC with fluorescence detection for the characterization of the bacterial diversity during cassava (Manihot esculenta Crantz) fermentation.
abstract: Denaturing high-performance liquid chromatography (DHPLC) has been described as a suitable method to study DNA polymorphisms. Here, cassava (Manihot esculenta Crantz) fermentation liquor was examined using DHPLC analysis to characterize the bacterial diversity during the fermentation process. GC-clamped amplicons corresponding to a variable region of the bacterial community 16S rDNA were synthesized using polymerase chain reaction (PCR) and then resolved on a base-composition basis using preparative DHPLC. Eluate fractions were collected at random and used as a source of whole community DNA that could be used to determine the bacterial diversity. As a first approach, GC-clamps were removed from the eluted DNA fragments using PCR to avoid the possible bias these clamps could cause during the construction of clone libraries. As a second approach, a clone library of each eluate sample was constructed, preserving the GC-clamps of the DNA fragments. The first approach generated 132 bacterial rDNA sequences with an average size of 200 bp, 45% of which had similarity to unculturable or non-classified bacteria. The second approach produced 194 sequences identified as Proteobacteria (48%), uncultured or non-classified environmental bacteria (40%) and Firmicutes (12%). We detected a remarkably greater bacterial diversity using the first approach than the second approach. The DHPLC-PCR method allowed for the fast and non-laborious detection of a vast bacterial diversity that was associated with cassava fermentation, and we conclude that it is a promising alternative for the characterization of the overall microbial diversity in complex samples.
authors.affiliation: Laboratório de Polimorfismo de DNA, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brasil.
authors.forename: C S
authors.initials: CS
authors.lastname: Kodama
authors.affiliation: Laboratório de Polimorfismo de DNA, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brasil.
authors.forename: S
authors.initials: S
authors.lastname: Cuadros-Orellana
authors.affiliation: Laboratório de Investigação Sistemática em Biotecnologia, Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Belém, PA, Brasil.
authors.forename: C H M M
authors.initials: CH
authors.lastname: Bandeira
authors.affiliation: Laboratório de Polimorfismo de DNA, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brasil.
authors.forename: D A
authors.initials: DA
authors.lastname: Graças
authors.affiliation: Laboratório de Investigação Sistemática em Biotecnologia, Instituto de Ciências Exatas e Naturais, Universidade Federal do Pará, Belém, PA, Brasil.
authors.forename: A S
authors.initials: AS
authors.lastname: Santos
authors.affiliation: Laboratório de Polimorfismo de DNA, Instituto de Ciências Biológicas, Universidade Federal do Pará, Belém, PA, Brasil asilva@ufpa.br.
authors.forename: A
authors.initials: A
authors.lastname: Silva
mesh_terms: D001419:Bacteria / Q000145:classification* / Q000235:genetics / Q000302:isolation & purification*; D002851:Chromatography, High Pressure Liquid; D003001:Cloning, Molecular; D015169:Colony Count, Microbial; D004269:DNA, Bacterial / Q000032:analysis; D004275:DNA, Ribosomal / Q000032:analysis; D005285:Fermentation; D002365:Manihot / Q000145:classification / Q000382:microbiology*; D054892:Metagenome; D064307:Microbiota; D008969:Molecular Sequence Data; D016133:Polymerase Chain Reaction; D017422:Sequence Analysis, DNA
chemical_list: D004269:DNA, Bacterial; D004275:DNA, Ribosomal