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.

Comparative Evaluation of Four Bacteria-Specific Primer Pairs for 16S rRNA Gene Surveys

“…Primer pair 341f/785r approximated best the qPCR data using group-specific qPCR primer sets (Pfeiffer et al, 2014), followed by 967f/1391r and 799f/1193r. Indeed, the ratio between the bacterial 16S rRNA gene copies as determined by qPCR supported the relative amount of bacterial phyla as estimated from the 454 amplicon library (Figure 5), indicating that 341f/785r used for the 454-sequencing allowed a high rate of coverage and amplified with relatively little bias under the applied conditions.…”

Environmental drivers of soil microbial community distribution at the Koiliaris Critical Zone Observatory

“…Understanding the drivers regulating the structure of soil microbial communities, their function and their activity comprise important challenges in the modern environmental microbiology. Pertinent information, even at high phylogenetic levels, has been useful to assign taxa with (aggregate) specific functions (Fierer et al, 2007;Philippot et al, 2009;Wess en et al, 2010), to associate broad microbial groups with certain ecosystem services (Six et al, 2006;Averill et al, 2014) and to provide support for a new generation of biogeochemical models explicitly addressing microbial structure (Waring et al, 2013).…”

“…The study of soil microbial biogeography is an emerging research field and lacks behind biogeochemical data and/or physical properties. Spatial studies in soil microbial community structure have been carried out at various scales, ranging from soil pore (Ruamps et al, 2011) to individual fields (Philippot et al, 2009), regional (Bru et al, 2011), country (Griffiths et al, 2011), continental level Fierer et al, 2013), or global level Nemergut et al, 2011;Serna-Chavez et al, 2013). The sampling density, the soil properties assessed (physical, chemical, biological), and the method and the depth of microbial community characterization diverge greatly among studies.…”

Secondary metabolite genes encoded by potato rhizosphere microbiomes in the Andean highlands are diverse and vary with sampling site and vegetation stage

“…From soils optimal concentrations of DNA were extracted by bead beating technique as described previously by Pfeiffer et al . 25 . Briefly, DNA was isolated from 0.5 g of each soil sample using FastDNA Spin Kit (MP Biomedicals) by bead beating thrice with phenol-chloroform-isoamyl alcohol (25:24:1).…”