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Clinical Veterinary Microbiology

Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica

Rickard Knutsson, Charlotta Löfström, Halfdan Grage, Jeffrey Hoorfar, Peter Rådström
Rickard Knutsson
1Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology
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Charlotta Löfström
1Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology
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Halfdan Grage
2Department of Mathematical Statistics, Lund University, Lund, Sweden
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Jeffrey Hoorfar
3Danish Veterinary Laboratory, Copenhagen, Denmark
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Peter Rådström
1Applied Microbiology, Center for Chemistry and Chemical Engineering, Lund Institute of Technology
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  • For correspondence: Peter.Radstrom@tmb.lth.se
DOI: 10.1128/JCM.40.1.52-60.2002
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    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.

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    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.
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    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 (▵).

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    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.

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  • TABLE 1.

    Experimental design and results from the factorial design experiment with the two PCR reaction mixtures based on AmpliTaq Gold and rTtha

    PCR mixtureFactorP value by ANOVAbExperimental designPCR results
    CTΔRnFactor levelConcnMean CTMean ΔRn
    AmpliTaq GoldMgCl20.0000.000High3.5 mM23.11.7
    Low1.5 mM36.20.2
    Center2.5 mM24.61.2
    Axial high4.7 mM22.31.9
    Axial low0.1 mM40.00.0
    Probe0.1920.050High125 nM30.61.1
    Low75 nM28.60.8
    Center100 nM26.21.2
    Axial high160 nM22.52.1
    Axial low40 nM24.50.6
    Primer0.2600.089High1,200 nM30.90.9
    Low600 nM28.31.0
    Center900 nM26.31.1
    Axial high1,600 nM23.31.4
    Axial low185 nM23.71.2
    AmpliTaq Gold0.2180.536High0.07 U/μl29.11.0
    Low0.03 U/μl30.10.9
    Center0.05 U/μl27.41.1
    Axial high0.10 U/μl23.61.4
    Axial low0.02 U/μl24.51.2
    DNA0.0000.474High5.0 × 10−8 g27.41.0
    Low5.0 × 10−10 g31.90.9
    Central5.0 × 10−9 g26.21.2
    Axial high2.0 × 10−6 g15.71.2
    Axial low2.0 × 10−11 g31.51.1
    rTthMgCl20.2350.082High3.5 mM24.55.5
    Low1.5 mM35.00.5
    Center2.5 mM22.35.7
    Axial high4.7 mM21.97.5
    Axial low0.1 mM36.80.1
    Probe0.6820.415High125 nM31.32.8
    Low75 nM28.22.8
    Center100 nM23.25.5
    Axial high160 nM22.44.1
    Axial low40 nM24.12.3
    Primer0.6810.204High1,200 nM28.13.2
    Low600 nM31.42.5
    Center900 nM23.45.0
    Axial high1,600 nM22.35.8
    Axial low185 nM22.08.6
    rTth0.7580.769High0.035 U/μl29.63.2
    Low0.015 U/μl29.82.4
    Central0.025 U/μl23.65.8
    Axial high0.043 U/μl23.13.6
    Axial low0.007 U/μl21.52.5
    DNA0.0030.015High5.0 × 10−8 g27.13.4
    Low5.0 × 10−10 g32.42.3
    Central5.0 × 10−9 g23.35.1
    Axial high2.0 × 10−6 g14.59.1
    Axial low2.0 × 10−11 g30.53.8
    • ↵ 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).

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Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
Rickard Knutsson, Charlotta Löfström, Halfdan Grage, Jeffrey Hoorfar, Peter Rådström
Journal of Clinical Microbiology Jan 2002, 40 (1) 52-60; DOI: 10.1128/JCM.40.1.52-60.2002

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Modeling of 5′ Nuclease Real-Time Responses for Optimization of a High-Throughput Enrichment PCR Procedure for Salmonella enterica
Rickard Knutsson, Charlotta Löfström, Halfdan Grage, Jeffrey Hoorfar, Peter Rådström
Journal of Clinical Microbiology Jan 2002, 40 (1) 52-60; DOI: 10.1128/JCM.40.1.52-60.2002
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KEYWORDS

Models, Biological
polymerase chain reaction
Salmonella enterica
Taq Polymerase

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