Evaluation of Homogenization Methods for Extraction of Live Bacteria and Recombinant DNA in Soil

Abstract

In many soil types, ubiquitous bacteria are present very in low quantities due to the inherent challenges of microorganism survival in environmental conditions. While, in some cases, only present in low quantity, soil microorganisms are a critical component of our ecosystem. Soil microbiome studies aim to characterize the microbial repertoire and to correlate the microbial population with soil and ecological functions. While many studies involve culturing and isolation of bacteria harvested from the soil, some organisms are hard to propagate in the laboratory or require the sample to be processed directly to maintain analyte integrity. Given time involved and complexity of culture based enrichments, many modern studies involve direct cell lysis and DNA recovery from soil followed by PCR for target enrichment and detection. In order to infer absolute taxa abundance non‐conserved, targeted gene products are quantified by qPCR. While this approach is rapid and robust, qPCR is subject to inhibitory effects of soil based compounds such as humic acids and polysaccharides.In this study, we compare the recovery and qPCR detection of a modified bacterial standard from multiple types of spiked soil samples. The standard was composed of E. coli, Bacillus subtilis, and Pseudomonas fluorescens which were genetically modified with green florescent protein (GFP). The modified organism allowed the absolute subtraction of background signal originating from native organisms found in the soil samples. The soil samples were spiked with decreasing quantities of the standard. The samples were extracted through a combination of bead milling and silica spin column based DNA purification. The DNA was then quantified by qPCR against the GFP gene and the resulting data established limits of recovery for the combination of organisms. These limits were evaluated side by side with a common incubation lysis to evaluate the effects of mechanical lysis on bacterial cells in soil samples that vary in resistance to lysis. Along with the limits of recovery, cycle threshold values (ct values) were compared to evaluate the impact of PCR inhibitor release.This abstract is from the Experimental Biology 2018 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.