Abstract
Quantitative-PCR (qPCR) enables the quantification of specific DNA targets, such as functional or phylogenetic marker genes associated with environmental samples. During each qPCR cycle, the number of copies of a gene (or region) of interest in DNA samples is determined in real time using a fluorescence-based label and compared to a standard serial dilution. Here, we describe a qPCR method to quantify the ammonia oxidizing bacteria involved in the first step of nitrification, using the amoA gene as a proxy of their abundance. The preparation of the standards from environmental samples and qPCR is presented in detail for specifically quantifying microbial abundance in environmental samples such as soil.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Smith CJ, Osborn AM (2009) Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67:6–20
Ririe KM, Rasmussen RP, Wittwer CT (1997) Product differentiation by analysis of DNA melting curves during the polymerase chain reaction. Anal Biochem 245:154–160
de Sosa LL, Glanville HC, Marshall MR et al (2018) Spatial zoning of microbial functions and plant-soil nitrogen dynamics across a riparian area in an extensively grazed livestock system. Soil Biol Biochem 120:153–164
Rotthauwe JH, Witzel KP, Liesack W (1997) The ammonia monooxygenase structural gene amoA as a functional marker: molecular fine-scale analysis of natural ammonia-oxidizing populations. Appl Environ Microbiol 63:4704–4712
Töwe S, Kleineidam K, Schloter M (2010) Differences in amplification efficiency of standard curves in quantitative real-time PCR assays and consequences for gene quantification in environmental samples. J Microbiol Methods 82:338–341
Dhanasekaran S, Doherty TM, Kenneth J (2010) Comparison of different standards for real-time PCR-based absolute quantification. J Immunol Methods 354:34–39
Bru D, Ramette A, Saby NPA et al (2011) Determinants of the distribution of nitrogen-cycling microbial communities at the landscape scale. ISME J 5:532–542
Bustin SA, Benes V, Garson JA et al (2009) The MIQE guidelines: minimum information for publication of quantitative real-time PCR experiments. Clin Chem 55:611–622
Smith CJ, Nedwell DB, Dong LF et al (2006) Evaluation of quantitative polymerase chain reaction-based approaches for determining gene copy and gene transcript numbers in environmental samples. Environ Microbiol 8:804–815
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2021 Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Blaud, A., Maïder, A., Clark, I.M. (2021). Quantification of Ammonia Oxidizing Bacterial Abundances in Environmental Samples by Quantitative-PCR. In: Carvalhais, L.C., Dennis, P.G. (eds) The Plant Microbiome. Methods in Molecular Biology, vol 2232. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-1040-4_12
Download citation
DOI: https://doi.org/10.1007/978-1-0716-1040-4_12
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-1039-8
Online ISBN: 978-1-0716-1040-4
eBook Packages: Springer Protocols