title: Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples.
abstract: PCR-based techniques are commonly used to characterize microbial communities, but are subject to bias that is difficult to assess. This study aimed to evaluate bias of several PCR primer-based strategies used to study diversity of autotrophic ammonia oxidizers. 16S rRNA genes from soil- or sediment-DNA were amplified using primers considered either selective or specific for betaproteobacterial ammonia oxidizers. Five approaches were assessed: (a) amplification with primers betaAMO143f-betaAMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with betaAMO143f-betaAMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with betaAMO143f-betaAMO1315r and CTO189f-Pf1053r primers; (e) nested amplification with 27f-1492r and CTO189f-CTO654r primers. Amplification products were characterized by denaturing gradient gel electrophoresis (DGGE) analysis after further amplification with 357f-GC-518r primers. DGGE profiles of soil communities were heterogeneous and depended on the approach followed. Ammonia oxidizer diversity was higher using approaches (b), (c) and (e) than using (a) and (d), where sequences of the most prominent bands showed similarities to nonammonia oxidizers. Profiles from marine sediments were more consistent, regardless of the approach adopted, and sequence analysis of excised bands indicated that these consisted of ammonia oxidizers only. The study demonstrates the importance of choice of primer, of screening for sequences of nontarget organisms and use of several approaches when characterizing microbial communities in natural environments.
authors.affiliation: School of Medical Sciences, Institute of Medical Sciences, University of Aberdeen, Foresterhill, Aberdeen, UK.
authors.forename: Shahid
authors.initials: S
authors.lastname: Mahmood
authors.forename: Thomas E
authors.initials: TE
authors.lastname: Freitag
authors.forename: James I
authors.initials: JI
authors.lastname: Prosser
mesh_terms: D000641:Ammonia / Q000378:metabolism*; D044822:Biodiversity; D004587:Electrophoresis, Agar Gel; D005784:Gene Amplification; D020459:Genes, rRNA / Q000235:genetics*; D009584:Nitrogen / Q000378:metabolism; D010084:Oxidation-Reduction; D016133:Polymerase Chain Reaction / Q000379:methods*; D012313:RNA / Q000235:genetics; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012623:Seawater / Q000382:microbiology; D017423:Sequence Analysis, RNA / Q000379:methods; D012988:Soil Microbiology
chemical_list: C034477:RNA primers; D012336:RNA, Ribosomal, 16S; D012313:RNA; D000641:Ammonia; D009584:Nitrogen