Quantification of Microorganisms Targeting Conserved Genes in Complex Environmental Samples Using qPCR
Claudia Goyer and Catherine E. Dandie
from: Quantitative Real-time PCR in Applied Microbiology (Edited by: Martin Filion). Caister Academic Press, U.K. (2012)
Development of quantitative PCR (qPCR) has facilitated major advances in assessment of microbial community abundances in complex environmental samples including water, soil, sediments, compost and manure and in our understanding of factors influencing community sizes in situ. qPCR has increasingly been used in environmental studies due to its sensitivity, ease of use, and the capacity to run large numbers of samples. However, qPCR has some limitations, which are specifically caused by the nature of environmental samples, including the variability in microorganism distribution, the efficiency of DNA recovery and purification, and the amount and type of PCR inhibitors co-extracted with the target nucleic acids. The heterogeneity of the templates amplified by qPCR can generate PCR biases and artifacts. Accuracy of the quantification of broad groups of microorganisms is influenced by the number of gene copies per genome of the selected marker. In this review, we will discuss the main experimental considerations for using qPCR in environmental studies, including the factors affecting key steps in the process of performing quantification of microorganisms in environmental samples. Although quantification of microorganisms is challenging, it is possible to reliably quantify microorganisms in complex environmental samples using qPCR. We will also briefly review the findings of studies which have used qPCR to quantify microorganisms from complex matrices read more ...