The near-quantitative sampling of genomic DNA from various food-borne Eubacteria

Abstract

BackgroundThe disruption of the bacterial cell wall plays an important part in achieving quantitative extraction of DNA from Eubacteria essential for accurate analyses of genetic material recovered from environmental samples.ResultsIn this work we have tested a dozen commercial bacterial genomic DNA extraction methodologies on an average of 7.70 × 106 (±9.05%), 4.77 × 108 (±31.0%), and 5.93 × 108 (±4.69%) colony forming units (CFU) associated with 3 cultures (n = 3) each of Brochothrix thermosphacta (Bt; Gram-positive), Shigella sonnei (Ss; Gram-negative), and Escherichia coli O79 (Ec; Gram-negative). We have utilized real-time PCR (qPCR) quantification with two specific sets of primers associated with the 16S rRNA “gene” to determine the number of copies CFU-1 by comparing the unknown target DNA qPCR results with standards for each primer set. Based upon statistical analyses of our results, we determined that the Agencourt Genfind v2, High Pure PCR Template Prep Kit, and Omnilyse methods consistently provided the best yield of genomic DNA ranging from 141 to 934, 8 to 21, and 16 to 27 16S rDNA copies CFU-1 for Bt, Ss, and Ec. If one assumes 6–7 copies of the 16S rRNA gene per genome, between 1 and 3 genomes per actively dividing cell and ≥ 100 cells CFU-1 for Bt (found to be a reasonable assumption using an optical method expounded upon herein) or between 1 and 2 cells CFU-1 for either Ss or Ec, then the Omnilyse procedure provided nearly quantitative extraction of genomic DNA from these isolates (934 ± 19.9 copies CFU-1 for Bt; 20.8 ± 2.68 copies CFU-1 for Ss; 26.9 ± 3.39 copies CFU-1 for Ec). The Agencourt, High Pure, and Omnilyse technologies were subsequently assessed using 5 additional Gram-positive and 10 Gram-negative foodborne isolates (n = 3) using a set of “universal” 16S rDNA primers.ConclusionOverall, the most notable DNA extraction method was found to be the Omnilyse procedure which is a “bead blender” technology involving high frequency agitation in the presence of zirconium silicate beads.Electronic supplementary materialThe online version of this article (doi:10.1186/s12866-014-0326-z) contains supplementary material, which is available to authorized users.

Nucleic Acid Extraction from Synthetic Mars Analog Soils forin situLife Detection

“…Current environmental analog testing is focused on optimizing extraction protocols with Atacama/MDV-like microbial populations in the synthetic Mars analog soils and environmental samples from Mars-relevant sites. The OmniLyse cell-lysis component of the PureLyse Bacterial gDNA Extraction Kits has previously demonstrated the ability to lyse down to 50 spores of B. subtilis and has been characterized as the “best yield” extraction method by the United States Department of Agriculture in comparison to 11 other extraction methods (Irwin et al, 2014 ). Prior validation of OmniLyse suggests that DNA extraction of low spore populations analogous to Mars-like environments is feasible.…”

Offline Next Generation Metagenomics Sequence Analysis Using MinION Detection Software (MINDS)

“…Effective lysis has been proven across a variety of cell types including gram-positive bacteria, spores, yeast, and cysts. Only 1–2 min is required to provide consistent yields of gDNA [23]. The small footprint of the OmniLyse and battery powered bead beating mechanism allows for easy use inside a glove box or biosafety cabinet when working with unknown, potentially high threat organisms.…”

Microbial community responses to soil tillage and crop rotation in a corn/soybean agroecosystem

“…The number of 16S rRNA copies in each sample was quantified using qPCR relative to Escherichia coli plate counts (Irwin et al., ). The number of colony‐forming units (CFU) of E. coli strain K‐12 (Carolina Biological Supply) was quantified using serial dilutions (plates were duplicated and the average used in calculations); there were 9.1 × 10 8 CFU/ml.DNA from the same E. coli was then isolated using a MO‐BIO UltraClean Microbial DNA isolation kit; an average of 2.6 μg of DNA was extracted from duplicate extractions.…”