Evaluation of PCR primer selectivity and phylogenetic specificity by using amplification of 16S rRNA genes from betaproteobacterial ammonia-oxidizing bacteria in environmental samples.
The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (betaAOB) was evaluated. betaAOB represent a group of phylogenetically related organisms for which the 16S rRNA gene approach is especially suitable. We used experimental comparisons of primer performance with water samples, together with an in silico analysis of published sequences and a literature review of clone libraries made with four specific PCR primers for the betaAOB 16S rRNA gene. With four aquatic samples, the primers NitA/NitB produced the highest frequency of ammonia-oxidizing-bacterium-like sequences compared to clone libraries with products amplified with the primer combinations betaAMOf/betaAMOr, betaAMOf/Nso1255g, and NitA/Nso1225g. Both the experimental examination of ammonia-oxidizing-bacterium-specific 16S rRNA gene primers and the literature search showed that neither specificity nor sensitivity of primer combinations can be evaluated reliably only by sequence comparison. Apparently, the combination of sequence comparison and experimental data is the best approach to detect possible biases of PCR primers. Although this study focused on betaAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.
Junier
Pilar
P
Ecole Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland. pilar.junier@epfl.ch
Kim
Ok-Sun
OS
Hadas
Ora
O
Imhoff
Johannes F
JF
Witzel
Karl-Paul
KP
D000641:Ammonia / Q000378:metabolism*; D020562:Betaproteobacteria / Q000235:genetics* / Q000378:metabolism; D017931:DNA Primers; D004269:DNA, Bacterial / Q000235:genetics*; D015723:Gene Library; D020459:Genes, rRNA / Q000235:genetics; D014644:Genetic Variation; D010084:Oxidation-Reduction; D010802:Phylogeny; D016133:Polymerase Chain Reaction; D012336:RNA, Ribosomal, 16S / Q000235:genetics*; D012680:Sensitivity and Specificity; D017422:Sequence Analysis, DNA; D014871:Water Microbiology
D017931:DNA Primers; D004269:DNA, Bacterial; D012336:RNA, Ribosomal, 16S; D000641:Ammonia