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Comparison among amoA primers suited for quantification and diversity analyses of ammonia-oxidizing bacteria in soil.

ABSTRACT: Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental samples using real-time PCR. We found that PCR-DGGE analysis with the amoA-2IR primer gave the best results in this studied soil. These results indicate that amoA-2IR is a suitable primer for community analysis of AOB in the environment.

SUBMITTER: Shimomura Y

PROVIDER: S-EPMC4036032 | biostudies-literature | 2012

REPOSITORIES: biostudies-literature

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Comparison among amoA primers suited for quantification and diversity analyses of ammonia-oxidizing bacteria in soil.

Shimomura Yumi Y Morimoto Sho S Takada Hoshino Yuko Y Uchida Yoshitaka Y Akiyama Hiroko H Hayatsu Masahito M

Microbes and environments 20111110 1

Ammonia monooxygenase subunit A gene (amoA) is frequently used as a functional gene marker for diversity analysis of ammonia-oxidizing bacteria (AOB). To select a suitable amoA primer for real-time PCR and PCR-denaturing gradient gel electrophoresis (DGGE), three reverse primers (degenerate primer amoA-2R; non-degenerate primers amoA-2R-GG and amoA-2IR) were examined. No significant differences were observed among the three primers in terms of quantitative values of amoA from environmental sampl ...[more]

PMID: 22075625

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Project description: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.

Project description:Black soils (Mollisols) of northeast China are highly productive and agriculturally important for food production. Ammonia-oxidizing microbes play an important role in N cycling in the black soils. However, the information related to the composition and distribution of ammonia-oxidizing microbes in the black soils has not yet been addressed. In this study, we used the amoA gene to quantify the abundance and community composition of ammonia-oxidizing archaea (AOA) and ammonia-oxidizing bacteria (AOB) across the black soil zone. The amoA abundance of AOA was remarkably larger than that of AOB, with ratios of AOA/AOB in the range from 3.1 to 91.0 across all soil samples. The abundance of AOA amoA was positively correlated with total soil C content (p < 0.001) but not with soil pH (p > 0.05). In contrast, the abundance of AOB amoA positively correlated with soil pH (p = 0.009) but not with total soil C. Alpha diversity of AOA did not correlate with any soil parameter, however, alpha diversity of AOB was affected by multiple soil factors, such as soil pH, total P, N, and C, available K content, and soil water content. Canonical correspondence analysis indicated that the AOA community was mainly affected by the sampling latitude, followed by soil pH, total P and C; while the AOB community was mainly determined by soil pH, as well as total P, C and N, water content, and sampling latitude, which highlighted that the AOA community was more geographically distributed in the black soil zone of northeast China than AOB community. In addition, the pairwise analyses showed that the potential nitrification rate (PNR) was not correlated with alpha diversity but weakly positively with the abundance of the AOA community (p = 0.048), whereas PNR significantly correlated positively with the richness (p = 0.003), diversity (p = 0.001) and abundance (p < 0.001) of the AOB community, which suggested that AOB community might make a greater contribution to nitrification than AOA community in the black soils when ammonium is readily available.

Dataset Information

Comparison among amoA primers suited for quantification and diversity analyses of ammonia-oxidizing bacteria in soil.

Publications

Comparison among amoA primers suited for quantification and diversity analyses of ammonia-oxidizing bacteria in soil.

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