Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples

Shahid Mahmood, Thomas Erich Freitag, James Ivor Prosser

Research output: Contribution to journalArticle

43 Citations (Scopus)

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 beta AMO143f-beta AMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with beta AMO143f-beta AMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with beta AMO143f-beta AMO1315r 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.

Original languageEnglish
Pages (from-to)482-493
Number of pages11
JournalFEMS Microbiology Ecology
Volume56
DOIs
Publication statusPublished - 2006

Keywords

  • 16S rRNA gene
  • denaturing gradient gel electrophoresis
  • ammonia oxidizing bacteria
  • grassland soil
  • marine sediment
  • 16S RIBOSOMAL-RNA
  • GRADIENT GEL-ELECTROPHORESIS
  • MOLECULAR ANALYSIS
  • GENE-SEQUENCES
  • MICROBIAL DIVERSITY
  • MARINE-SEDIMENTS
  • BACTERIA
  • DNA
  • SOIL
  • BIAS

Cite this

Comparison of PCR primer-based strategies for characterization of ammonia oxidizer communities in environmental samples. / Mahmood, Shahid; Freitag, Thomas Erich; Prosser, James Ivor.

In: FEMS Microbiology Ecology, Vol. 56, 2006, p. 482-493.

Research output: Contribution to journalArticle

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AB - 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 beta AMO143f-beta AMO1315r; (b) amplification with primers CTO189f-CTO654r; (c) nested amplification with beta AMO143f-beta AMO1315r followed by CTO189f-CTO654r primers; (d) nested amplification with beta AMO143f-beta AMO1315r 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.

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KW - BACTERIA

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JO - FEMS Microbiology Ecology

JF - FEMS Microbiology Ecology

SN - 1574-6941

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