Quantitative real-time PCR (qPCR) to estimate molecular fungal abundance

Abstract

Fungi are key players in the decomposition of leaves in freshwaters. This functional role is maintained by a specifically adapted fungal community. To assess the quantitative contribution of single fungal species to the process, it is essential to determine their abundance. Quantitative real-time PCR (qPCR) is the prevalent method for this purpose, because it detects individual species of aquatic fungi in samples composed of multiple species. Quantitative PCR reactions are an extension of the traditional PCR method, which facilitates measuring the exponential amplification of a specific gene region via the emission of fluorescence signals in real time. This chapter describes how to design and validate a qPCR assay for fungal litter decomposers. The method uses a taxon-specific Taqman® probe labelled with a fluorescent reporter which hybridizes between two PCR primers. Due to the 5'-3'-exonuclease activity of DNA polymerase during PCR, the reporter dye is released and the emitted fluorescence is measured at 465-510 nm. Monitoring fungal taxa by qPCR assays opens excellent opportunities to gain new insights in microbial community ecology and ecosystem processes such as litter decomposition that are driven by fungi. © Springer Nature Switzerland AG 2020