Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR

Ângela Lima; Lúcia G V Sousa; Nuno Cerca

doi:10.1007/978-1-0716-3358-8_9

Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR

Methods Mol Biol. 2023:2967:105-115. doi: 10.1007/978-1-0716-3358-8_9.

Authors

Ângela Lima^{1

2}, Lúcia G V Sousa¹, Nuno Cerca^{3

4}

Affiliations

¹ Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal.
² LABBELS -Associate Laboratory, Braga, Guimarães, Portugal.
³ Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), CEB - Centre of Biological Engineering, University of Minho, Braga, Portugal. nunocerca@ceb.uminho.pt.
⁴ LABBELS -Associate Laboratory, Braga, Guimarães, Portugal. nunocerca@ceb.uminho.pt.

PMID: 37608106
DOI: 10.1007/978-1-0716-3358-8_9

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.

Keywords: Bacterial populations; Calibration curves; DNA extraction; Exogenous control; Reaction efficiency; qPCR.

Publication types

Research Support, Non-U.S. Gov't

MeSH terms

Bacteria* / genetics
Calibration
DNA, Bacterial / genetics
Genome, Bacterial
Polymerase Chain Reaction* / methods

Substances

DNA, Bacterial