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BMC Microbiol. 2014 Dec 31;14:326. doi: 10.1186/s12866-014-0326-z.

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

Author information

1
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. Peter.Irwin@ars.usda.gov.
2
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. Ly.Nguyen@ars.usda.gov.
3
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. Yiping.He@ars.usda.gov.
4
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. George.Paoli@ars.usda.gov.
5
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. Andrew.Gehring@ars.usda.gov.
6
Agricultural Research Service, U. S. Department of Agriculture, Molecular Characterization of Foodborne Pathogens Research Unit, Eastern Regional Research Center, 600 East Mermaid Lane, Wyndmoor, PA, 19038, USA. Chin-Yi.Chen@ars.usda.gov.

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 × 10(6) (±9.05%), 4.77 × 10(8) (±31.0%), and 5.93 × 10(8) (±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.

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.

PMID:
25551371
PMCID:
PMC4302497
DOI:
10.1186/s12866-014-0326-z
[Indexed for MEDLINE]
Free PMC Article

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