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Front. Microbiol. | doi: 10.3389/fmicb.2019.00036

Design and assessment of species-level qPCR primers targeting comammox

  • 1Civil and Environmental Engineering, College of Engineering, University of Wisconsin-Madison, United States

Published PCR primers targeting the ammonia monooxygenase gene (amoA) were applied to samples from activated sludge systems operated with low dissolved oxygen (DO) to quantify total and clade-level Nitrospira that perform complete ammonium oxidation (comammox); however, we found these existing primers resulted in significant artifact-associated non-target amplification. This not only overestimated comammox amoA copies but also resulted in numerous false positive detections in the environmental samples tested, as confirmed by gel electrophoresis. Therefore, instead of attempting to quantify comammox diversity, we focused on accurately quantifying the candidate comammox species. We designed specific and sensitive primers targeting 3 candidate species: Candidatus (Ca.) Nitrospira nitrosa, Ca. N. inopinata, and Ca. N. nitrificans. The primers were tested with amoA templates of these candidate species and used to quantify comammox at the species level in low DO activated sludge systems. We found that comammox related to Ca. N. nitrosa were present and abundant in the majority of samples from low DO bioreactors and were not detected in samples from a high DO system. In addition, the greatest abundance of Ca. N. nitrosa was found in bioreactors operated with a long solids retention time. Ca. N. inopinata and Ca. N. nitrificans were only detected sporadically in these samples, indicating a minor role of these comammox in nitrification under low DO conditions.

Keywords: Comammox, Nitrospira, Nitrification, Low dissolved oxygen, biological nutrient removal, qPCR, Real time PCR (qPCR), PCR primer design

Received: 09 Jul 2018; Accepted: 10 Jan 2019.

Edited by:

Simona Rossetti, Istituto di ricerca sulle acque (IRSA), Italy

Reviewed by:

Seung Gu Shin, Pohang University of Science and Technology, South Korea
Luciano Beneduce, Dipartimento di Scienze Agrarie, degli Alimenti e dell'Ambiente, Università degli Studi di Foggia, Italy  

Copyright: © 2019 Keene-Beach and Noguera. This is an open-access article distributed under the terms of the Creative Commons Attribution License (CC BY). The use, distribution or reproduction in other forums is permitted, provided the original author(s) and the copyright owner(s) are credited and that the original publication in this journal is cited, in accordance with accepted academic practice. No use, distribution or reproduction is permitted which does not comply with these terms.

* Correspondence: Prof. Daniel R. Noguera, College of Engineering, University of Wisconsin-Madison, Civil and Environmental Engineering, Madison, 53706, South Dakota, United States,