title: Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa.
abstract: Mapping the distribution of phylogenetically distinct bacteria in natural environments is of primary importance to an understanding of ecological dynamics. Here we present a quantitative PCR (qPCR) assay for the analysis of higher taxa composition in natural communities that advances previously available methods by allowing quantification of several taxa during the same qPCR run. Existing primers targeting the 16S rRNA gene specific for Firmicutes, Actinobacteria, Bacteroidetes and for the α and γ subdivisions of the Proteobacteria were improved by largely increasing the coverage of the taxon they target without diminishing their specificity. The qPCR assay was validated in vitro testing artificial mixtures of 16S rRNA sequences and used to characterise the composition of natural communities developing in young marine biofilms. The possible contribution of the proposed technique in revealing ecological dynamics affecting higher bacterial taxa is discussed.
authors.affiliation: School of Marine Science and Technology, Ridley Building, Newcastle University, Newcastle upon Tyne NE1 7RU, England, UK.
authors.forename: Tristano
authors.initials: T
authors.lastname: Bacchetti De Gregoris
authors.forename: Nick
authors.initials: N
authors.lastname: Aldred
authors.forename: Anthony S
authors.initials: AS
authors.lastname: Clare
authors.forename: J Grant
authors.initials: JG
authors.lastname: Burgess
mesh_terms: D039903:Actinobacteria / Q000145:classification / Q000235:genetics; D059001:Aquatic Organisms / Q000145:classification* / Q000235:genetics; D015373:Bacterial Typing Techniques / Q000379:methods*; D041963:Bacteroidetes / Q000145:classification / Q000235:genetics; D001483:Base Sequence; D018441:Biofilms / Q000145:classification*; D017931:DNA Primers / Q000235:genetics*; D004269:DNA, Bacterial / Q000032:analysis* / Q000235:genetics; D010802:Phylogeny; D020560:Proteobacteria / Q000145:classification / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000032:analysis / Q000235:genetics; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods*; D017422:Sequence Analysis, DNA
chemical_list: D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S