Skip to main content
. 2016 Nov 9;82(23):6930–6941. doi: 10.1128/AEM.01613-16

TABLE 6.

DNA recovery and qPCR using pellet or supernatant obtained by glass bead decomposition of heat-killed E. coli with transmembrane proteins in the cell wall exposed to the Pd or fixation agenta

Source DNA extraction Treatment for LD discriminationd E. coli boiled in distilled sterile water (2.8 × 109 cells/ml)
OD260/OD280 DNA concn (ng/μl) CT by qPCR (cycle) (5 ng/qPCR)
Pelletb SAV/PPR/SDS + P/C/I Fixation 1.57 ± 0.06 105.7 ± 4.41 31.1 ± 0.41
Pd (1,000 μM) 1.64 ± 0.06 132.3 ± 6.62 27.3 ± 0.38
NT (no treatment) 1.88 ± 0.04 217.5 ± 8.95 20.1 ± 0.64
SDS + P/C/I Fixation 1.61 ± 0.04 12.0 ± 0.51 ND
Pd (1,000 μM) 1.62 ± 0.02 8.4 ± 0.42 38.1 ± 0.68
NT (no treatment) 1.63 ± 0.04 188.1 ± 11.13 20.3 ± 0.65
P/C/I alone Fixation 1.55 ± 0.03 8.4 ± 0.54 ND
Pd (1,000 μM) 1.52 ± 0.01 4.1 ± 0.42 ND
NT (no treatment) 1.61 ± 0.01 108.5 ± 3.47 21.8 ± 0.58
Supernatantc SAV/PPR/SDS + P/C/I Fixation 1.71 ± 0.09 10.5 ± 0.62 37.1 ± 0.48
Pd (1,000 μM) 1.74 ± 0.07 6.1 ± 0.55 35.5 ± 0.63
NT (no treatment) 1.66 ± 0.04 61.8 ± 4.35 25.7 ± 0.54
SDS + P/C/I Fixation 1.53 ± 0.06 3.4 ± 0.41 ND
Pd (1,000 μM) 1.68 ± 0.05 4.2 ± 0.56 40.8 ± 0.71
NT (no treatment) 1.60 ± 0.08 55.3 ± 3.34 25.9 ± 0.55
P/C/I alone Fixation 1.48 ± 0.04 2.2 ± 0.36 ND
Pd (1,000 μM) 1.62 ± 0.03 3.6 ± 0.34 ND
NT (no treatment) 1.56 ± 0.04 35.6 ± 2.84 24.6 ± 0.74
a

Heat-killed E. coli cells were decomposed with glass beads. SAV/PPR/SDS: savinase, peptidase R, and SDS were added. P/C/I: DNA extraction by phenol, chloroform, and isoamyl alcohol. Pd: Cl2(ŋ-cycloocta-1,5-diene)Pd(II). ND, no qPCR elongation of the target.

b

A 10 mM concentration of Tris-HCl was added to decomposed E. coli fragments followed by centrifugation to produce the pellet. In detail, the pellet obtained by the centrifugation was supplied for DNA extraction.

c

The supernatant obtained by the centrifugation of decomposed E. coli fragments was applied for DNA extraction.

d

Mildform 10NM was used to cross-link chromosomal DNA with transmembrane proteins in E. coli cells for fixation.