Accurate Absolute Quantification of Bacterial Populations in Mixed Cultures by qPCR

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.