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Design and Assessment of Species-Level qPCR Primers Targeting Comammox

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 prime...

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Autores principales: Beach, Natalie K., Noguera, Daniel R.
Formato: Online Artículo Texto
Lenguaje:English
Publicado: Frontiers Media S.A. 2019
Materias:
Acceso en línea:https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365651/
https://www.ncbi.nlm.nih.gov/pubmed/30766515
http://dx.doi.org/10.3389/fmicb.2019.00036
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author Beach, Natalie K.
Noguera, Daniel R.
author_facet Beach, Natalie K.
Noguera, Daniel R.
author_sort Beach, Natalie K.
collection PubMed
description 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.
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spelling pubmed-63656512019-02-14 Design and Assessment of Species-Level qPCR Primers Targeting Comammox Beach, Natalie K. Noguera, Daniel R. Front Microbiol Microbiology 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. Frontiers Media S.A. 2019-01-31 /pmc/articles/PMC6365651/ /pubmed/30766515 http://dx.doi.org/10.3389/fmicb.2019.00036 Text en Copyright © 2019 Beach and Noguera. http://creativecommons.org/licenses/by/4.0/ 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.
spellingShingle Microbiology
Beach, Natalie K.
Noguera, Daniel R.
Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title_full Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title_fullStr Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title_full_unstemmed Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title_short Design and Assessment of Species-Level qPCR Primers Targeting Comammox
title_sort design and assessment of species-level qpcr primers targeting comammox
topic Microbiology
url https://www.ncbi.nlm.nih.gov/pmc/articles/PMC6365651/
https://www.ncbi.nlm.nih.gov/pubmed/30766515
http://dx.doi.org/10.3389/fmicb.2019.00036
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