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State-specific extraction of environmental DNA: Spike-and-recovery controls to validate and optimize extraction protocols – Universität Innsbruck

Seminar of the Department of Microbiology

State-specific extraction of environmental DNA: Spike-and-recovery controls to validate and optimize extraction protocols

Julia Zöhrer – PhD candidate - UIBK - Bodenmikrobiologie und Ökophysiologie 

07.11.2024, 11:00 - Hybrid

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Abstract

Julia Zöhrer

The direct extraction and analysis of the total environmental DNA (eDNA) enables the routine study of microbial communities at large scale and high taxonomic resolution. However, eDNA consists of both extracellular DNA (exDNA) and intracellular DNA (iDNA) revealing even more profound insights into the diversity of currently prevalent communities and/or past assemblages. Starting in the 1980s, a multitude of methodological approaches for the separation of exDNA and iDNA from environmental samples has been published. Even though many of them are conceptually similar, both methodological biases and related consequences for the interpretation of eDNA-based studies are poorly evaluated due to the lack of appropriate experimental setups. Based on single-gene deletion mutants of Escherichia coli and Bacillus subtilis, we developed spike-and-recovery controls for the extraction of eDNA accounting for both the different eDNA states (iDNA, exDNA) as well as their bacterial origin (gram-negative, gram-positive). Unique primer/probe sets were designed for each spike-in and therefore allowed the absolute quantification by multiplex digital PCR (dPCR), which was proven successful within different environments such as soil, sediment, sludge from anaerobic digestion and compost. Hence, the proposed approach provides a valuable step towards the simplified application of spike-and-recovery controls and highlights its potential to better understand the methodological ambiguities related to the separation of exDNA and iDNA, facilitating the validation and further optimization of such protocols.

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