1887

Abstract

The gene encodes nitrous oxide reductase, a key enzyme in the nitrous oxide reduction that occurs during complete denitrification. Many conventional approaches have used Proteobacteria-based primers to detect in environmental samples. However, these primers often fail to detect in non-Proteobacteria strains, including Firmicutes (Gram-positive) and Bacteroidetes. In this study, newly designed primers successfully amplified this gene from five species (Firmicutes). The primers were used to construct clone libraries from DNA extracted from sludge and domestic animal feedlot soils, all with high organic carbon contents. After DNA sequencing, phylogenetic analysis identified many new sequences with high levels of homology to from Bacteroidetes, probably because of the high sequence similarity of from Firmicutes and Bacteroidetes, and a predominance of Bacteroidetes in feedlot environments. Three sets of new quantitative real-time PCR (qPCR) primers based on our clone library sequences were designed and tested for their specificities. Our data showed that only Bacteroidetes-related sequences were amplified, whereas conventional Proteobacteria-based primers amplified only Proteobacteria-related . Quantitative analysis of with the new qPCR primers recovered ~10 copies per 100 ng DNA. Thus, it appears that amplification with conventional primers is insufficient for developing an understanding of the diversity and abundance of genes in the environment.

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2013-02-01
2019-12-11
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