Abstract
After the discovery of complete ammonia-oxidizing (comammox) Nitrospira, detection and assessments of the contribution of comammox Nitrospira communities to nitrogen cycling are in great demand. PCR-based approach, a common method for the detection of comammox, depends strongly on accurate amplification of the amoA genes from the original DNA samples using appropriate primers. In this study, we reported an evaluation of the performance of two commonly used primer sets, Ntsp-amoA 162F/359R and comaA/B-244f/659r, for amplifying the comammox amoA genes from three representative wetland soils in China [Sangsang (SS), Sanjiang (SJ), and Xianghai (XH)]. Our results demonstrated the two primer sets could both successfully amplify the clades with high relative abundances (RA), and further revealed a broadly similar diversity and community composition of dominant comammox operational taxonomic units (OTUs) (RA ≥ 1%) in each of the three wetland soils. However, the clades with low RA, such as the clade A (1.26%) in SJ and the clade B (11.54%) in XH that were recovered by metagenomics analysis, failed to be amplified using comaA/B-244f/659r, but were successfully amplified and sequenced using Ntsp-amoA 162F/359R. It indicated that, compared to comaA/B-244f/659r, Ntsp-amoA 162F/359R was more sensitive to the clades with low RA. However, it is worth noting that Ntsp-amoA 162F/359R would overestimate the RA of some rare clades. For example, the RAs of clade B in XH were overestimated by 32-fold. Furthermore, high levels of non-target amplification were detected via gel electrophoresis using both primer sets, especially for comammox Clade B amoA genes, implying that we should treat qPCR results based on these primers with caution. Taken together, our study comprehensively compared the performance of the two primer sets on the sensitivity and specificity of amplifying comammox amoA genes in three wetland soils, pointing out the necessity of further development of new primers for the efficient and accurate detection of comammox in various environments.
Highlights
Nitrification, the aerobic oxidation of ammonia via nitrite to nitrate, is an essential part of the global nitrogen (N) cycling process (Galloway et al, 2008)
In order to compare the efficiency of two primer sets in amplifying comammox amoA genes and their ability to reveal the diversity of comammox community, PCR and agarose gel electrophoresis were conducted with the two primer sets for three typical wetland soils from the Tibetan Plateau and the Northeast Plain of China
Regarding to the primer set comaA/B-244f/659r, comammox clade A amoA genes were detected in SS and XH wetland soils with high specificity (Figure 2B)
Summary
Nitrification, the aerobic oxidation of ammonia via nitrite to nitrate, is an essential part of the global nitrogen (N) cycling process (Galloway et al, 2008). It was originally considered to be a two-step process catalyzed by two distinct functional groups of microorganisms, the ammonia-oxidizing microbes (AOM) including ammonia-oxidizing bacteria (AOB) and ammoniaoxidizing archaea (AOA) (Könneke et al, 2005), and nitriteoxidizing bacteria (NOB). This perception was overturned in 2015 with the surprising discovery of complete ammonia oxidizers (comammox) (Daims et al, 2015; van Kessel et al, 2015), which encode all the necessary enzymes involved in ammonia oxidation and nitrite oxidation in their genomes and perform complete nitrification activity in a single organism (Santoro, 2016). Current knowledge about the ecological distribution of phylotypes and their potential contribution to nitrification is still limited
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