Article Figures & Data
Figures
- FIG. 1.
Graphical illustration of Salmonella DNA amplification kinetics of the 5′ nuclease PCR assay (13) for the low and high axial levels used in the factorial design experiment. In all five graphs (A to E) the highest final ΔRn was obtained for the high axial concentrations of each PCR reagent. All PCR reagents except the PCR reagent being studied are at the concentration of the center level (Table 1). (A) High (4.7 mM) and low (0.1 mM) MgCl2 concentrations; (B) high (160 nM) and low (40 nM) probe concentrations; (C) high (1,600 nM) and low (185 nM) primer (both forward and reverse primers) concentrations; (D) high (0.10 U/μl) and low (0.02 U/μl) AmpliTaq Gold concentrations; (E) high (2 × 10−6 g/microwell) and low (2 × 10−11 g/microwell) DNA concentrations.
- FIG. 2.
Graphical appearance of the model used to estimate PCR performance. The experiment was performed three times with 10-fold dilutions of DNA, and the model (the line connecting the datum points) fits the experimental data (*) that were obtained well. Salmonella DNA amplification took place in the presence of ddH2O and different media with two amplification mixtures: (A) AmpliTaq Gold with ddH2O; (B) rTth with ddH2O; (C) AmpliTaq Gold with BPW; (D) rTth with BPW; (E) AmpliTaq Gold with BHI; (F) rTth with BHI. The vertical dashed lines in all graphs are the log DNA concentration at a detection probability of 0.95 from the estimated model. The horizontal dashed lines at CT equal to 30 marks the proposed upper limit for sufficient PCR performance. From the model the slope was determined and all graphs (A to F) gave close to optimal amplification efficiencies.
- FIG. 3.
DNA amplification by the Salmonella 5′ nuclease PCR assay in the presence of 5 μl of BHI and 5 μl of BPW. The graphs show the results of the CT response at a constant Salmonella DNA concentration (2 × 10−9 g/microwell) while the concentrations of BHI and BPW in the media were changed. The experiment was performed with both the AmpliTaq Gold mixture and the rTth mixture. The PCR results correspond to the following DNA amplification combinations: AmpliTaq Gold mixture and BPW medium (⧫), AmpliTaq Gold mixture and BHI medium (▴), rTth mixture and BPW medium (⋄), and rTth mixture and BHI medium (▵).
- FIG. 4.
Enrichment PCR for S. enterica serovar Enteritidis. The graph illustrates the dynamic detection range, i.e., the time during which positive detection is possible, by plotting the CT value against the incubation time. BPW was inoculated with S. enterica serovar Enteritidis at a concentration of 1 CFU/ml, and the mixture was incubated at 37°C. Samples for PCR analysis were withdrawn every 4 h. •, numbers of CFU per milliliter; ▪, results for AmpliTaq Gold mixture; ▴, results for rTth mixture.
Tables
- TABLE 1.
Experimental design and results from the factorial design experiment with the two PCR reaction mixtures based on AmpliTaq Gold and rTtha
PCR mixture Factor P value by ANOVAb Experimental design PCR results CT ΔRn Factor level Concn Mean CT Mean ΔRn AmpliTaq Gold MgCl2 0.000 0.000 High 3.5 mM 23.1 1.7 Low 1.5 mM 36.2 0.2 Center 2.5 mM 24.6 1.2 Axial high 4.7 mM 22.3 1.9 Axial low 0.1 mM 40.0 0.0 Probe 0.192 0.050 High 125 nM 30.6 1.1 Low 75 nM 28.6 0.8 Center 100 nM 26.2 1.2 Axial high 160 nM 22.5 2.1 Axial low 40 nM 24.5 0.6 Primer 0.260 0.089 High 1,200 nM 30.9 0.9 Low 600 nM 28.3 1.0 Center 900 nM 26.3 1.1 Axial high 1,600 nM 23.3 1.4 Axial low 185 nM 23.7 1.2 AmpliTaq Gold 0.218 0.536 High 0.07 U/μl 29.1 1.0 Low 0.03 U/μl 30.1 0.9 Center 0.05 U/μl 27.4 1.1 Axial high 0.10 U/μl 23.6 1.4 Axial low 0.02 U/μl 24.5 1.2 DNA 0.000 0.474 High 5.0 × 10−8 g 27.4 1.0 Low 5.0 × 10−10 g 31.9 0.9 Central 5.0 × 10−9 g 26.2 1.2 Axial high 2.0 × 10−6 g 15.7 1.2 Axial low 2.0 × 10−11 g 31.5 1.1 rTth MgCl2 0.235 0.082 High 3.5 mM 24.5 5.5 Low 1.5 mM 35.0 0.5 Center 2.5 mM 22.3 5.7 Axial high 4.7 mM 21.9 7.5 Axial low 0.1 mM 36.8 0.1 Probe 0.682 0.415 High 125 nM 31.3 2.8 Low 75 nM 28.2 2.8 Center 100 nM 23.2 5.5 Axial high 160 nM 22.4 4.1 Axial low 40 nM 24.1 2.3 Primer 0.681 0.204 High 1,200 nM 28.1 3.2 Low 600 nM 31.4 2.5 Center 900 nM 23.4 5.0 Axial high 1,600 nM 22.3 5.8 Axial low 185 nM 22.0 8.6 rTth 0.758 0.769 High 0.035 U/μl 29.6 3.2 Low 0.015 U/μl 29.8 2.4 Central 0.025 U/μl 23.6 5.8 Axial high 0.043 U/μl 23.1 3.6 Axial low 0.007 U/μl 21.5 2.5 DNA 0.003 0.015 High 5.0 × 10−8 g 27.1 3.4 Low 5.0 × 10−10 g 32.4 2.3 Central 5.0 × 10−9 g 23.3 5.1 Axial high 2.0 × 10−6 g 14.5 9.1 Axial low 2.0 × 10−11 g 30.5 3.8 ↵ a The different factors and their concentrations are given. The experiment with the AmpliTaq Gold mixture used a basic 25 factorial design with 2-by-5 axial points and one observation at the center point, giving a total of 86 observations. The experiment with the rTth mixture used a basic 25 factorial design augmented with 2-by-5 axial points and five observations at the center point, giving a total of 47 observations.
↵ b ANOVA, one-way analysis of variance for main effects in studying the significance of the different factors. Significant factor interactions are not presented here but are given in the text (see Results and Discussion).
