The near-quantitative sampling of genomic DNA from various food-borne EubacteriaReport as inadecuate




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BMC Microbiology

, 14:326

Applied microbiology

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 × 10 ±9.05%, 4.77 × 10 ±31.0%, and 5.93 × 10 ±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 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 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 for Bt found to be a reasonable assumption using an optical method expounded upon herein or between 1 and 2 cells CFU for either Ss or Ec, then the Omnilyse procedure provided nearly quantitative extraction of genomic DNA from these isolates 934 ± 19.9 copies CFU for Bt; 20.8 ± 2.68 copies CFU for Ss; 26.9 ± 3.39 copies CFU 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.

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.

Abbreviations, symbols, and equationsBt =Brochothrix thermosphacta ground chicken isolate 1

Ss =Shigella sonnei ground chicken isolate 1

Ec =Escherichia coli O79 whole chicken carcass isolate

O-type determined 2 July 2013 by the E. coli Reference CenterThe Pennsylvania State University, University Park, PA 16802

Rn = normalized fluorescence signal with respect to cycle number C which is typically sigmoidal in shape i.e.∂Rn-∂C has a near-Gaussian line-shape

C∂i or j =extrapolated cycle number where ∂Rn-∂C = 0 for any i or j dilution

Ti =i dilution of the standard target gene copies μL solution being amplified; Ti = 0 = 1.31 × 10 16S r DNA copies μL Bt standard used for all Gram-positive organisms, 1.06 × 10 copies μL Ss standard used for all Gram-negative organisms except Ec, or 9.66 × 10 copies μL Ec standard

∂ C ∂ i ∂ L o g 10 T i Open image in new window=change in C∂i with Log10Ti ideally ∂C∂i-∂LogβTi = −Log2β; ∂C∂i-∂LogβTi is always equivalent to ∂C∂i-∂Logβϕ and ϕ is the dilution factor; in this work ϕ = β and β, the base of the logarithm, is always 10 25

∂ C ∂ j ∂ L o g 10 0.1 j Open image in new window =slope of C∂j with respect to Log100.1 i.e., dilutions of a DNA extract of unknown concentration, j = 0, 1, 2, or 3

C∂intobs = intercept calculated from linear regression analysis of C∂i as affected by changes in Log10Ti ideally C∂intobs = C∂i + Log2Ti 25

C∂intpredicted j= C ∂ j − ∂ C ∂ j ∂ L o g 10 0.1 j × L o g 10 1 + ε i C ∂ int o b s − C ∂ j Open image in new window; predicted intercept for each junk dilution; derivation of C∂intpredicted jwas fully developed elsewhere 25

C ¯ ∂ int predicted Open image in new window =C∂intpredicted j averaged across all j

εstnd =Taq DNA polymerase efficiency associated with standard dilutions = − 1 + 10 − ∂ C ∂ i - ∂ L o g 10 T i − 1 Open image in new window

εunk =Taq DNA polymerase efficiency associated with unknown dilutions = − 1 + 10 − ∂ C ∂ j - ∂ L o g 10 ϕ j − 1 Open image in new window; poor εunk s e.g. 0.9 ≥ εunk ≥ 1.1 are possible indicators of enzyme perturbation 26 by inhibitory substances in an extract

Ŧj =traditional calculation of the unknown target gene DNA concentration copies μL of extract for the j dilution = 1 + ε stnd C ∂ int o b s – C ∂ j Open image in new window

Tj = j dilution of the corrected unknown DNA concentration copies μL of extract = 1 + ε u n k C ¯ ∂ int predicted – C ∂ j Open image in new window; this calculation corrects 25 for the fact that εstnd sometimes is substantially different than εunk and is the value reported in all Tables; when εstnd ~ εunkŦj ~ Tj

δ = organism concentration or density CFU mL r RNA -gene- copies CFU = Tj=0 in units of copies μL of extract × total μL of extract ÷ CFUs in 1 mL of culture; the values of the total assay volume have been provided at the end of each extraction procedure listed below. The average value x ¯ Open image in new window of each biological replicate’s CFU mL are listed in all Tables ± coefficients of variation CV = s ÷ x ¯ Open image in new window. Since all counting-based data have technical replicate variances ~ x ¯ Open image in new window assuming the number of observations-dilution were appropriately high, we report x ¯ Open image in new window and CV of the CFU mL calculated from 2 or three 1:10 dilutions of the starting concentration.

EE =extraction efficiency = observed 16S r DNA copies CFU÷ 16S r RNA gene copies genome × genomes cell × cells CFU÷ plating efficiency; e.g.; assuming 24 copies CFU÷ 7 copies genome × 1.5 genomes cell × 1.5 cells CFU÷ 0.67 plating efficiency 67%, would result in a near 100% efficiency; gene copies genome can vary between 1 and 14 but typically between 5 and 7; genomes cell would probably vary between 1 and 3 but is dependent upon the rate of cell division cells CFU varies greatly depending on the organism but typically ranges between 1 and 2 for Ss, Ec, and Salmonella spp.; plating efficiency is a correction for losses on solid media e.g., for organisms like Ss and Ec, this term could vary between 50 and 100%

SS = sum of squares

TMS= treatment mean square

-=Treatment SS-m-1 -

EMSE = Eerror mean square -=Total SS – Block SS + Treatment SS-m-1*n-1-

F = Fstatistic -=TMS-EMS-

-=AVERAGExk 1׃xkn-

SE= experimental standard error -=SQRTEMS-n-

P = the probability of rejecting the null hypothesis when it is true i.e., when there is no relationship between two measured observations; means were characteristically taken to be -significantly different- when P ≤ 0.05

qP = the -Studentized- range distribution tabulated in numerous statistics texts 27, 28 for a P = 0.01 or 0.05

tP =-Student’s t- at probability P - =TINVPn-2-

Electronic supplementary materialThe online version of this article doi:10.1186-s12866-014-0326-z contains supplementary material, which is available to authorized users.

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Author: Peter Irwin - Ly Nguyen - Yiping He - George Paoli - Andrew Gehring - Chin-Yi Chen

Source: https://link.springer.com/







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