Improvement of phylum- and class-specific primers for real-time PCR quantification of bacterial taxa

• Published in: Journal of microbiological methods Vol. 86; no. 3; pp. 351 - 356
• Main Authors: Bacchetti De Gregoris, Tristano, Aldred, Nick, Clare, Anthony S., Burgess, J. Grant
• Format: Journal Article
• Language: English
• Published: Amsterdam Elsevier B.V 01-09-2011; Elsevier
• Subjects: Actinobacteria; Actinobacteria - classification; Actinobacteria - genetics; Aquatic Organisms - classification; Aquatic Organisms - genetics; Bacteria; Bacterial Typing Techniques - methods; Bacteroidetes - classification; Bacteroidetes - genetics; Base Sequence; biofilm; Biofilms - classification; Biological and medical sciences; Broad taxonomic unit; DNA Primers - genetics; DNA, Bacterial - analysis; DNA, Bacterial - genetics; Firmicutes; Fundamental and applied biological sciences. Psychology; genes; in vitro studies; Microbiology; nucleotide sequences; Phylogeny; Proteobacteria; Proteobacteria - classification; Proteobacteria - genetics; qPCR; quantitative polymerase chain reaction; Real-Time Polymerase Chain Reaction - methods; ribosomal RNA; RNA, Ribosomal, 16S - analysis; RNA, Ribosomal, 16S - genetics; Sequence Analysis, DNA; SYBR green; Bacteria; qPCR; SYBR green; Broad taxonomic unit; Polymerase chain reaction; Real time; Quantitative analysis
• ISSN: 0167-7012; 1872-8359
• DOI: 10.1016/j.mimet.2011.06.010

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.
► Primers targeting higher bacterial taxa were designed. ► The use of these primers in qPCR improves previously available methods. ► The qPCR protocol presented was tested on artificial and natural communities. ► Marine biofilms showed reproducible changes in higher taxa composition.