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

Content Provider	Springer Nature : BioMed Central
Author	Irwin, Peter Nguyen, Ly He, Yiping Paoli, George Gehring, Andrew Chen, Chin-Yi
Abstract	Background The 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. Results In 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 (q PCR) quantification with two specific sets of primers associated with the 16S r RNA “gene” to determine the number of copies CFU-1 by comparing the unknown target DNA q PCR 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 r DNA copies CFU-1 for Bt, Ss, and Ec. If one assumes 6–7 copies of the 16S r RNA 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 r DNA primers. Conclusion Overall, 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.
Related Links	https://bmcmicrobiol.biomedcentral.com/counter/pdf/10.1186/s12866-014-0326-z.pdf
Ending Page	17
Page Count	17
Starting Page	1
File Format	HTM / HTML
ISSN	14712180
DOI	10.1186/s12866-014-0326-z
Journal	BMC Microbiology
Issue Number	1
Volume Number	14
Language	English
Publisher	BioMed Central
Publisher Date	2014-12-31
Access Restriction	Open
Subject Keyword	Microbiology Biological Microscopy Mycology Parasitology Virology Life Sciences Colony Form Unit Much Probable Number Bacterial Pellet Copy Number Data Assay Volume
Content Type	Text
Resource Type	Article
Subject	Microbiology Microbiology (medical)
Journal Impact Factor	4/2023
5-Year Journal Impact Factor	4.6/2023

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1	Ministry of Education (GoI), Department of Higher Education	Sanctioning Authority	https://www.education.gov.in/ict-initiatives
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