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Applied and Environmental Microbiology, August 2008, p. 5231-5236, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00288-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.

Evaluation of PCR Primer Selectivity and Phylogenetic Specificity by Using Amplification of 16S rRNA Genes from Betaproteobacterial Ammonia-Oxidizing Bacteria in Environmental Samples{triangledown} ,{dagger}

Pilar Junier,1,2* Ok-Sun Kim,2,3 Ora Hadas,4 Johannes F. Imhoff,5 and Karl-Paul Witzel2

École Polytechnique Fédérale de Lausanne, CH-1015 Lausanne, Switzerland,1 Max Planck Institute for Evolutionary Biology, 24306 Ploen, Germany,2 School of Biological Sciences and Institute of Microbiology, Seoul National University, 56-1 Shillim-dong, Kwanak-gu, Seoul 151-742, Republic of Korea,3 Israel Oceanographic and Limnological Research, The Kinneret Limnological Laboratory, 14950 Migdal, Israel,4 Leibniz Institute of Marine Sciences at the University of Kiel, 24105 Kiel, Germany5

Received 3 February 2008/ Accepted 8 June 2008

The effect of primer specificity for studying the diversity of ammonia-oxidizing betaproteobacteria (βAOB) was evaluated. βAOB 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 βAOB 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 βAMOf/βAMOr, βAMOf/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 βAOB, the results presented here more generally exemplify the importance of primer selection and potential primer bias when analyzing microbial communities in environmental samples.

* Corresponding author. Mailing address: EPFL ENAC ISTE EML, CE 1 644 (Centre Est), Station 6, CH-1015 Lausanne, Switzerland. Phone: 41 21 693 63 96. Fax: 41 21 693 62 05. E-mail: pilar.junier{at}epfl.ch

{triangledown} Published ahead of print on 20 June 2008.

{dagger} Supplemental material for this article may be found at http://aem.asm.org/.

Applied and Environmental Microbiology, August 2008, p. 5231-5236, Vol. 74, No. 16
0099-2240/08/$08.00+0     doi:10.1128/AEM.00288-08
Copyright © 2008, American Society for Microbiology. All Rights Reserved.





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