Dataset Information

Design and Assessment of Species-Level qPCR Primers Targeting Comammox.

ABSTRACT: 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.

SUBMITTER: Beach NK

PROVIDER: S-EPMC6365651 | biostudies-literature | 2019

REPOSITORIES: biostudies-literature

ACCESS DATA

Publications

Design and Assessment of Species-Level qPCR Primers Targeting Comammox.

Beach Natalie K NK Noguera Daniel R DR

Frontiers in microbiology 20190131

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 i ...[more]

PMID: 30766515

Similar Datasets

Project description:Background:Quantitative real-time PCR (qPCR) is a well-established method for detecting and quantifying bacteria, and it is progressively replacing culture-based diagnostic methods in food microbiology. High-throughput qPCR using microfluidics brings further advantages by providing faster results, decreasing the costs per sample and reducing errors due to automatic distribution of samples and reagents. In order to develop a high-throughput qPCR approach for the rapid and cost-efficient quantification of microbial species in complex systems such as fermented foods (for instance, cheese), the preliminary setup of qPCR assays working efficiently under identical PCR conditions is required. Identification of target-specific nucleotide sequences and design of specific primers are the most challenging steps in this process. To date, most available tools for primer design require either laborious manual manipulation or high-performance computing systems. Results:We developed the SpeciesPrimer pipeline for automated high-throughput screening of species-specific target regions and the design of dedicated primers. Using SpeciesPrimer, specific primers were designed for four bacterial species of importance in cheese quality control, namely Enterococcus faecium, Enterococcus faecalis, Pediococcus acidilactici and Pediococcus pentosaceus. Selected primers were first evaluated in silico and subsequently in vitro using DNA from pure cultures of a variety of strains found in dairy products. Specific qPCR assays were developed and validated, satisfying the criteria of inclusivity, exclusivity and amplification efficiencies. Conclusion:In this work, we present the SpeciesPrimer pipeline, a tool to design species-specific primers for the detection and quantification of bacterial species. We use SpeciesPrimer to design qPCR assays for four bacterial species and describe a workflow to evaluate the designed primers. SpeciesPrimer facilitates efficient primer design for species-specific quantification, paving the way for a fast and accurate quantitative investigation of microbial communities.

Dataset Information

Design and Assessment of Species-Level qPCR Primers Targeting Comammox.

Publications

Design and Assessment of Species-Level qPCR Primers Targeting Comammox.

Similar Datasets

OmicsDI is part of the ELIXIR infrastructure

Tweets