title: Improved group-specific primers based on the full  SILVA 16S rRNA gene reference database.
abstract: Quantitative PCR (qPCR) and community fingerprinting methods, such as the Terminal Restriction Fragment Length Polymorphism (T-RFLP) analysis,are well-suited techniques for the examination of microbial community structures. The use of phylum and class-specific primers can provide enhanced sensitivity and phylogenetic resolution as compared with domain-specific primers. To date, several phylum- and class-specific primers targeting the 16S ribosomal RNA gene have been published. However, many of these primers exhibit low discriminatory power against non-target bacteria in PCR. In this study, we evaluated the precision of certain published primers in silico and via specific PCR. We designed new qPCR and T-RFLP primer pairs (for the classes Alphaproteobacteria and Betaproteobacteria, and the phyla Bacteroidetes, Firmicutes and Actinobacteria) by combining the sequence information from a public dataset (SILVA SSU Ref 102 NR) with manual primer design. We evaluated the primer pairs via PCR using isolates of the above-mentioned groups and via screening of clone libraries from environmental soil samples and human faecal samples. As observed through theoretical and practical evaluation, the primers developed in this study showed a higher level of precision than previously published primers, thus allowing a deeper insight into microbial community dynamics.
authors.forename: Stefan
authors.initials: S
authors.lastname: Pfeiffer
authors.forename: Milica
authors.initials: M
authors.lastname: Pastar
authors.forename: Birgit
authors.initials: B
authors.lastname: Mitter
authors.forename: Kathrin
authors.initials: K
authors.lastname: Lippert
authors.forename: Evelyn
authors.initials: E
authors.lastname: Hackl
authors.forename: Paul
authors.initials: P
authors.lastname: Lojan
authors.forename: Andreas
authors.initials: A
authors.lastname: Oswald
authors.forename: Angela
authors.initials: A
authors.lastname: Sessitsch
mesh_terms: D001419:Bacteria / Q000145:classification / Q000235:genetics*; D001483:Base Sequence; D003198:Computer Simulation; D017931:DNA Primers / Q000737:chemistry* / Q000235:genetics; D030541:Databases, Genetic; D005243:Feces / Q000382:microbiology; D020459:Genes, rRNA; D006801:Humans; D008969:Molecular Sequence Data; D010802:Phylogeny; D016133:Polymerase Chain Reaction / Q000379:methods; D012150:Polymorphism, Restriction Fragment Length; D012336:RNA, Ribosomal, 16S / Q000235:genetics; D012988:Soil Microbiology
chemical_list: D017931:DNA Primers; D012336:RNA, Ribosomal, 16S