Abstract
Quantitative PCR (qPCR) is a well-established technique that allows to accurately quantify nucleic acids or proteins, being widely used in several types of biological samples for bacterial load quantification. However, there are many recent studies that do not consider the potential pitfalls involved in key experimental qPCR stages, namely, those related to the extraction and purification of genomic DNA and to the thermal amplification process, that can lead to biased results in mixed cultures. Herein, we outline a proper protocol for bacterial quantification by qPCR, addressing how to overcome the main issues in that methodology.
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
Similar content being viewed by others
References
Benes V, Castoldi M (2010) Expression profiling of microRNA using real-time quantitative PCR, how to use it and what is available. Methods 50:244–249. https://doi.org/10.1016/j.ymeth.2010.01.026
Yu J, Wang HM, Zha MS et al (2015) Molecular identification and quantification of lactic acid bacteria in traditional fermented dairy foods of Russia. J Dairy Sci 98:5143–5154. https://doi.org/10.3168/jds.2015-9460
Rocha D, Santos C, Pacheco L (2015) Bacterial reference genes for gene expression studies by RT-qPCR: survey and analysis. Antonie Van Leeuwenhoek 108:685–693. https://doi.org/10.1007/s10482-015-0524-1
Ståhlberg A, Thomsen C, Ruff D, Åman P (2012) Quantitative PCR analysis of DNA, RNAs, and proteins in the same single cell. Clin Chem 58:1682–1692. https://doi.org/10.1373/clinchem.2012.191445
Smith CJ, Osborn AM (2009) Advantages and limitations of quantitative PCR (Q-PCR)-based approaches in microbial ecology. FEMS Microbiol Ecol 67:6–20. https://doi.org/10.1111/j.1574-6941.2008.00629.x
Bustin SA (2010) Why the need for qPCR publication guidelines ? – the case for MIQE. Methods 50:217–226. https://doi.org/10.1016/j.ymeth.2009.12.006
Cotto A, Looper JK, Mota LC, Son A (2015) Quantitative polymerase chain reaction for microbial growth kinetics of mixed culture system. J Microbiol Biotechnol 25:1928–1935. https://doi.org/10.4014/jmb.1503.03090
Leigh Greathouse K, Sinha R, Vogtmann E (2019) DNA extraction for human microbiome studies: the issue of standardization. Genome Biol 20:1–4. https://doi.org/10.1186/s13059-019-1843-8
Costea PI, Zeller G, Sunagawa S et al (2017) Towards standards for human fecal sample processing in metagenomic studies. Nat Biotechnol 35:1069–1076. https://doi.org/10.1038/NBT.3960
Magalhães AP, França Â, Pereira MO, Cerca N (2019) RNA-based qPCR as a tool to quantify and to characterize dual-species biofilms. Sci Rep 9:1–12. https://doi.org/10.1038/s41598-019-50094-3
Marotz C, Amir A, Humphrey G et al (2017) DNA extraction for streamlined metagenomics of diverse environmental samples. BioTechniques 62:290–293. https://doi.org/10.2144/000114559
O’Connell GC, Chantler PD, Barr TL (2017) High interspecimen variability in nucleic acid extraction efficiency necessitates the use of spike-in control for accurate qPCR-based measurement of plasma cell-free DNA levels. Lab Med 48:332–338. https://doi.org/10.1093/LABMED/LMX043
Cerca N, Lima Â, França A (2022) Accurate qPCR quantification in polymicrobial communities requires assessment of gDNA extraction efficiency. J Microbiol Methods 194:1–4. https://doi.org/10.1016/j.mimet.2022.106421
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. https://doi.org/10.1373/CLINCHEM.2008.112797
Svec D, Tichopad A, Novosadova V et al (2015) How good is a PCR efficiency estimate: recommendations for precise and robust qPCR efficiency assessments. Biomol Detect Quantif 3:9–16. https://doi.org/10.1016/j.bdq.2015.01.005
Longin C, Guilloux-benatier M, Alexandre H (2016) Design and performance testing of a DNA extraction assay for sensitive and reliable quantification of acetic acid bacteria directly in red wine using real time PCR. Front Microbiol 7:1–9. https://doi.org/10.3389/fmicb.2016.00831
Rosca A, Castro J, Sousa L et al (2022) In vitro interactions within a biofilm containing three species found in bacterial vaginosis (BV) support the higher antimicrobial tolerance associated with BV recurrence. J Antimicrob Chemother 77:2183–2190
Coyne KJ, Handy SM, Demir E et al (2005) Improved quantitative real-time PCR assays for enumeration of harmful algal species in field samples using an exogenous DNA reference standard. Limnol Oceanogr Methods 3:381–391. https://doi.org/10.4319/lom.2005.3.381
Taskin B, Gozen AG, Duran M (2011) Selective quantification of viable Escherichia coli bacteria in biosolids by quantitative PCR with propidium monoazide modification. Appl Environ Microbiol 77:4329–4335. https://doi.org/10.1128/AEM.02895-10
Scarsella E, Zecconi A, Cintio M, Stefanon B (2021) Characterization of microbiome on feces, blood and milk in dairy cows with different milk leucocyte pattern. Animals 11:1–14. https://doi.org/10.3390/ani11051463
Livak KJ, Schmittgen TD (2001) Analysis of relative gene expression data using real-time quantitative PCR and the 2-ΔΔCT method. Methods 25:402–408. https://doi.org/10.1006/meth.2001.1262
Acknowledgments
This work was funded by the Portuguese Foundation for Science and Technology (FCT) under the scope of the strategic funding of the unit (UIDB/04469/2020) and EXPL/SAU-INF/1000/2021. AL and LGVS are supported by FCT with individual grants 2022.13112.BD and 2020.04912.BD, respectively.
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2023 The Author(s), under exclusive license to Springer Science+Business Media, LLC, part of Springer Nature
About this protocol
Cite this protocol
Lima, Â., Sousa, L.G.V., Cerca, N. (2023). Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR. In: Domingues, L. (eds) PCR. Methods in Molecular Biology, vol 2967. Humana, New York, NY. https://doi.org/10.1007/978-1-0716-3358-8_9
Download citation
DOI: https://doi.org/10.1007/978-1-0716-3358-8_9
Published:
Publisher Name: Humana, New York, NY
Print ISBN: 978-1-0716-3357-1
Online ISBN: 978-1-0716-3358-8
eBook Packages: Springer Protocols