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Results: 5

1.
Figure 4

Figure 4. Ratiometric pre-rRNA analysis M. bovis BCG cells in serum.. From: Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix.

Cells were incubated separately in filtered and unfiltered human serum at 37°C for 30 days. The serum-incubated cells were then resuspended in pre-warmed 7H9 broth and samples were taken after 0,1, 2, 4, and 24 hours. P:G ratios were calculated as in Figure 2. Values are means and SDs of nine ratiometric permutations from three technical replicates of each sample type. From a five-point gDNA standard curve, qPCR efficiency was calculated as 1.001. A separate experiment with H37Ra yielded similar results (Figure S2).

Kris M. Weigel, et al. PLoS One. 2013;8(1):e54886.
2.
Figure 5

Figure 5. Ratiometric pre-rRNA analysis of A. baumannii cells in serum by using a rapid semi-automated approach.. From: Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix.

Serum-incubated cells were plated to quantify viable CFU/mL, then serially diluted in serum. Viable CFU/mL values (X-axis) were subsequently calculated from plating results. To initiate nutritional stimulation, dilutions in serum were further diluted 1∶10 into TSB or PBS (stimulated and non-stimulated, respectively). After 90 minutes, pre-rRNA was quantified by one-step probe-based q-RT-PCR as described in text. Values are means and SDs of ΔCt values (non-stimulated minus stimulated) from two replicates of each dilution. Control samples with no bacteria (0 CFU/mL) yielded no RT-qPCR results, and therefore could not be plotted as ΔCt values. Reaction efficiency could not be calculated for this experiment, because no standard curve was used. A replicate experiment (Figure S3) yielded similar results, showing a limit of detection of ≤56 viable CFU/mL.

Kris M. Weigel, et al. PLoS One. 2013;8(1):e54886.
3.
Figure 3

Figure 3. Ratiometric pre-rRNA analysis of A. baumannii (A), P. aeruginosa (B), and S. aureus (C) cells in serum over time.. From: Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix.

Three biological replicates for each organism were prepared at ∼1E5 CFU/mL in serum and analyzed after 4, 24, and 168 hours of serum acclimation. At each timepoint, nutritional stimulation was initiated by suspending cells in pre-warmed TSB for 1.5 hours. Changes in pre-rRNA are expressed as means and standard deviations of the fold-increases in P:G ratio following nutritional stimulation, relative to non-stimulated control aliquots (P:G+/P:G−). The horizontal dashed line indicates the “viability threshold” which samples with viable cells are expected to exceed. From separate gDNA standard curves consisting of ≥ five points each, qPCR efficiencies were between 1.010 and1.067.

Kris M. Weigel, et al. PLoS One. 2013;8(1):e54886.
4.
Figure 1

Figure 1. Reverse transcription and PCR primers.. From: Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix.

A: Oligonucleotides used for priming reverse transcription of pre-rRNA (cDNA synthesis), and forward and reverse PCR primers for amplifying cDNA. B: Specificity of PCR primer sets designed for S. aureus, A. baumannii, P. aeruginosa, and MTBC. Each primer set was used in endpoint PCR conducted on purified DNA from all the four organisms. Key: L, 1 Kb+ DNA ladder; 1, S. aureus DNA; 2, A. baumannii DNA; 3, P. aeruginosa DNA; 4, M. tuberculosis DNA; 5, negative control (water). Lowest three bands of ladder are 100, 200, and 300 bp. A repeat experiment yielded identical results.

Kris M. Weigel, et al. PLoS One. 2013;8(1):e54886.
5.
Figure 2

Figure 2. Ratiometric pre-rRNA analysis of A. baumannii, S. aureus, and P. aeruginosa cells in serum.. From: Molecular Viability Testing of Bacterial Pathogens from a Complex Human Sample Matrix.

A–C: Analysis of cells that had been held in serum for 7 days. Nutritional stimulation was initiated by suspending cells in pre-warmed TSB, and samples taken after 0, 1, 2, and 4 hours were subjected to RT-qPCR and qPCR to quantify pre-rRNA and gDNA, respectively. The same primers were used to amplify gDNA and cDNA generated from pre-rRNA. Ratios of pre-rRNA to gDNA (P:G; bars) are means and SDs of nine ratiometric permutations from three technical replicates of each sample type. Quantity of gDNA (lines) are means and standard deviations of the three gDNA measurements. Viable cell densities of A. baumannii, S. aureus, and P. aeruginosa, respectively, in serum were 9.0×108, 9.7×105, and <1×102 CFU/mL. From separate gDNA standard curves consisting of ≥ five points each, qPCR efficiencies were calculated [10(−1/slope) −1] to be between 0.913 and 0.959. A replicate experiment (Figure S1) yielded similar results for all three organisms.

Kris M. Weigel, et al. PLoS One. 2013;8(1):e54886.

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