PMID- 23505422
OWN - NLM
STAT- MEDLINE
DCOM- 20130906
LR  - 20211021
IS  - 1932-6203 (Electronic)
IS  - 1932-6203 (Linking)
VI  - 8
IP  - 3
DP  - 2013
TI  - Evaluating primers for profiling anaerobic ammonia oxidizing bacteria within 
      freshwater environments.
PG  - e57242
LID - 10.1371/journal.pone.0057242 [doi]
LID - e57242
AB  - Anaerobic ammonia oxidizing (anammox) bacteria play an important role in 
      transforming ammonium to nitrogen gas and contribute to fixed nitrogen losses in 
      freshwater environments. Understanding the diversity and abundance of anammox 
      bacteria requires reliable molecular tools, and these are not yet well 
      established for these important Planctomycetes. To help validate PCR primers for 
      the detection of anammox bacteria within freshwater ecosystems, we analyzed 
      representative positive controls and selected samples from Grand River and 
      groundwater sites, both from Ontario, Canada. The objectives of this study were 
      to identify a suitable anammox denaturing gradient gel electrophoresis (DGGE) 
      fingerprint method by using GC-clamp modifications to existing primers, and to 
      verify the specificity of anammox-specific primers used for DGGE, cloning and 
      qPCR methods. Six primer combinations were tested from four published primer sets 
      (i.e. A438f/A684r, Amx368f/Amx820r, An7f/An1388r, and Pla46/1392r) for both 
      direct and nested PCR amplifications. All PCR products were run subsequently on 
      DGGE gels to compare the resulting patterns. Two anammox-specific primer 
      combinations were also used to generate clone libraries and quantify anammox 
      bacterial 16S rRNA genes with qPCR. The primer set A438f/A684r was highly 
      specific to anammox bacteria, provided reliable DGGE fingerprints and generated a 
      high proportion of anammox-related clones. A second primer set (Amx368f/Amx820r) 
      was anammox specific, based on clone library analysis, but PCR products from 
      different candidate species of anammox bacteria resolved poorly using DGGE 
      analysis. Both DGGE and cloning results revealed that Ca. Brocadia and an 
      uncharacterized anammox bacterial cluster represented the majority of anammox 
      bacteria found in Grand River sediment and groundwater samples, respectively. 
      Together, our results demonstrate that although Amx368f/Amx820r was useful for 
      anammox-specific qPCR and clone library analysis, A438f/A684r was the most 
      suitable primer set for multiple molecular assessments of anammox bacteria in 
      freshwater environments.
FAU - Sonthiphand, Puntipar
AU  - Sonthiphand P
AD  - Department of Biology, University of Waterloo, Waterloo, Ontario, Canada.
FAU - Neufeld, Josh D
AU  - Neufeld JD
LA  - eng
SI  - GENBANK/JX392915
SI  - GENBANK/JX392916
SI  - GENBANK/JX392917
SI  - GENBANK/JX392918
SI  - GENBANK/JX392919
SI  - GENBANK/JX392920
SI  - GENBANK/JX392921
SI  - GENBANK/JX392922
SI  - GENBANK/JX392923
SI  - GENBANK/JX392924
SI  - GENBANK/JX392925
SI  - GENBANK/JX392926
SI  - GENBANK/JX392927
SI  - GENBANK/JX392928
SI  - GENBANK/JX392929
SI  - GENBANK/JX392930
SI  - GENBANK/JX392931
SI  - GENBANK/JX392932
SI  - GENBANK/JX392933
SI  - GENBANK/JX392934
SI  - GENBANK/JX392935
SI  - GENBANK/JX392936
SI  - GENBANK/JX392937
SI  - GENBANK/JX392938
SI  - GENBANK/JX392939
SI  - GENBANK/JX392940
SI  - GENBANK/JX392941
SI  - GENBANK/JX392942
SI  - GENBANK/JX392943
SI  - GENBANK/JX392944
SI  - GENBANK/JX392945
SI  - GENBANK/JX392946
SI  - GENBANK/JX392947
SI  - GENBANK/JX392948
PT  - Journal Article
PT  - Research Support, Non-U.S. Gov't
DEP - 20130307
PL  - United States
TA  - PLoS One
JT  - PloS one
JID - 101285081
RN  - 0 (DNA Primers)
RN  - 0 (DNA, Bacterial)
RN  - 0 (RNA, Ribosomal, 16S)
RN  - 7664-41-7 (Ammonia)
SB  - IM
MH  - Ammonia/*metabolism
MH  - Bacteria, Anaerobic/classification/*genetics/*metabolism
MH  - Biodiversity
MH  - DNA Primers
MH  - DNA, Bacterial
MH  - Ecosystem
MH  - Electrophoresis, Polyacrylamide Gel/methods
MH  - Fresh Water/*microbiology
MH  - *Gene Expression Profiling
MH  - Molecular Sequence Data
MH  - Ontario
MH  - Oxidation-Reduction
MH  - Phylogeny
MH  - RNA, Ribosomal, 16S
PMC - PMC3591393
COIS- Competing Interests: The authors have declared that no competing interests exist.
EDAT- 2013/03/19 06:00
MHDA- 2013/09/07 06:00
PMCR- 2013/03/07
CRDT- 2013/03/19 06:00
PHST- 2012/08/03 00:00 [received]
PHST- 2013/01/18 00:00 [accepted]
PHST- 2013/03/19 06:00 [entrez]
PHST- 2013/03/19 06:00 [pubmed]
PHST- 2013/09/07 06:00 [medline]
PHST- 2013/03/07 00:00 [pmc-release]
AID - PONE-D-12-23477 [pii]
AID - 10.1371/journal.pone.0057242 [doi]
PST - ppublish
SO  - PLoS One. 2013;8(3):e57242. doi: 10.1371/journal.pone.0057242. Epub 2013 Mar 7.