Accurate qPCR quantification in polymicrobial communities requires assessment of gDNA extraction efficiency

Nuno Cerca; Ângela Lima; Angela França

doi:10.1016/j.mimet.2022.106421

Accurate qPCR quantification in polymicrobial communities requires assessment of gDNA extraction efficiency

J Microbiol Methods. 2022 Mar:194:106421. doi: 10.1016/j.mimet.2022.106421. Epub 2022 Feb 1.

Authors

Nuno Cerca¹, Ângela Lima², Angela França²

Affiliations

¹ Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; LABBELS -Associate Laboratory, Braga, Guimarães, Portugal. Electronic address: nunocerca@ceb.uminho.pt.
² Laboratory of Research in Biofilms Rosário Oliveira (LIBRO), Centre of Biological Engineering (CEB), University of Minho, Campus de Gualtar, 4710-057 Braga, Portugal; LABBELS -Associate Laboratory, Braga, Guimarães, Portugal.

PMID: 35114291
DOI: 10.1016/j.mimet.2022.106421

Abstract

qPCR absolute quantification requires the assessment of PCR reaction efficiency. This is achieved by performing serial dilutions of gDNA. Herein, we demonstrate that when quantifying bacterial load in mixed samples, reaction efficiency should not be used as a calibration curve since gDNA isolation efficiency is neglected, significantly impacting quantification.

Keywords: Bacterial vaginosis; Polimicrobial communities; gDNA extraction efficiency; gDNA qPCR bacterial quantification.

Publication types

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

MeSH terms

Bacterial Load
DNA*
Real-Time Polymerase Chain Reaction

Substances

DNA