Research ArticleDIAGNOSTICS

Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

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Science Advances  04 Sep 2015:
Vol. 1, no. 8, e1400061
DOI: 10.1126/sciadv.1400061
  • Fig. 1 The DOTS qPCR device.

    (A) The DOTS qPCR device showing all components: semicircular channel with PID-controlled heaters positioned at both ends, feedback thermocouples mounted 5 mm above the heater surface, motor arm with looped thermocouple mounted for droplet suspension in heated oil, viewing window at the center of the channel, and lens tube to focus and magnify the droplet image onto the smartphone camera. All components are disposable after one use except for the motor, lens tube, and smartphone. (B) Alternate view showing the DOT moving to the low-temperature side of the heat gradient. (C) Two thermocouples mounted on the motor arm. The straight thermocouple is used for oil temperature measurement, and the looped thermocouple holds the droplet and measures the droplet temperature. (D and E) Still images of the submerged droplet moving back and forth continuously at the low-temperature region. The thermocouple junction is inside the droplet to monitor the reaction temperature. Droplet temperature feedback is used by the motor program to accurately position the droplet in the heat gradient. The still images are screen captures from the supplementary video showing device operation (movie S3). (F) Still image of the droplet moving away from the low-temperature region after completing annealing, to be positioned at a warmer region corresponding to the optimum temperature for Taq polymerase extension of the PCR amplicon.

  • Fig. 2 Thermal characteristics and reproducibility of device.

    (A) Temperature color map of the heat gradient established between heaters with a maximum of 100°C on the left and a minimum of 50°C on the right. (B) Heat ramping of the two extreme temperature regions from 25°C to equilibrium within 10 min. (C) Representative thermocycling profile of the internal droplet temperature and surrounding oil temperature. Desired temperatures are consistently achieved even at sub-minute cycle times. The temperatures at each phase are 90.4° ± 0.2°C for denaturation, 68.4° ± 0.2°C for extension, and 60.2° ± 0.2°C for annealing. Droplet ramp rates up to 12°C/s and oil ramp rates up to 32°C/s are achieved by moving the droplet within the heat gradient. (D) Gel electropherogram showing the results from three successive trials (lanes 1 to 3) to amplify the 196-bp 16S rRNA V3 amplicon from 7 pg of purified K. pneumoniae genomic DNA (equivalent to 1.4 × 103 genomic copies) and an NTC sample. The thermocycling speed was 48 s/cycle, and 30 cycles were conducted. The band intensities in lanes 1 to 3 have a coefficient of variation of 4.0%.

  • Fig. 3 Interfacial tension and fluorescence qPCR inhibition of the IE model.

    (A) Protein concentrations of the aortic, mitral, and tricuspid valve sections excised from a porcine heart and ground using a micro–mortar and pestle. The total protein concentration of the tissue model is 1.6 ± 0.1 mg/ml. (B) The interfacial tensions (γ) of clean and contaminated PCR mixtures are 25.55 and 27.60 mN/m, respectively. (C) Free-body force diagram with the interfacial layer. The forces on the droplet include the interfacial tension force (Fγ), the buoyancy force (FB), the weight of the droplet (Fmg), and the thermocouple force (FTC). (D) Fluorescence qPCR amplification curves for 16S rRNA hypervariable regions V1-V2 and vanA gene from intact vancomycin-resistant E. faecium (VRE) with and without tissue contamination. The Ct values for 16S rRNA V1-V2 without tissue, 16S rRNA V1-V2 with tissue, vanA without tissue, and vanA with tissue are 28.4, 30.0, 34.0, and 39.4, respectively. The tissue contamination inhibits fluorescence qPCR, as seen by the upward shift of 1.6 cycles for the 16S rRNA V1-V2 target and 5.4 cycles for the vanA target. Additionally, NTC samples for each primer set are plotted. (E) Protein diffusion to the interface is calculated on the basis of typical blood and tissue concentrations, using diffusivities from literature and Fick’s equation. For comparison, the diffusion of Taq polymerase to the interface is also calculated. (F and G) The porcine heart from which heart valves were excised, sectioned, inoculated, ground, and used as the PCR target.

  • Fig. 4 Specificity, limit of detection, and speed of DOT thermocycling.

    (A) Gel electropherogram showing the differentiation of vancomycin-resistant E. faecium (VRE) and vancomycin-sensitive E. faecalis (VSE) by multiplexed amplification of the 377-bp vanA amplicon directly from bacterial culture. Simultaneous thermocycling was achieved by mounting three droplets on different thermocouples on the same motor arm. Lane 1, 1-kb Plus DNA Ladder; lane 2, VRE; lane 3, VSE; lane 4, NTC; lane 5, 1-kb Plus DNA Ladder. (B) Gel electropherogram showing the limit of detection at the sub-picogram level by amplification of the 196-bp 16S rRNA V3 amplicon from 0.7 pg of K. pneumoniae genomic DNA (equivalent to 1.4 × 102 genomic copies) at a speed of 48 s/cycle. Lane 1, 1-kb Plus DNA Ladder; lane 2, 0.7-pg genomic DNA. (C) Gel electropherogram showing rapid amplification of the 16S rRNA V3 amplicon and vanA amplicon in the presence of tissue contaminants in 30 cycles. Lane 1, vanA amplified at 40 s/cycle (20 min) from 7 × 105 CFU VRE inoculated to valve tissue (V3 amplified from 7 × 105 CFU VRE inoculated to valve tissue); lane 2, at 40 s/cycle (20 min); lane 3, at 32 s/cycle (16 min); lane 4, at 28 s/cycle (14 min); lane 5, 1-kb Plus DNA Ladder (see Fig. 2D for V3 NTC results).

  • Fig. 5 Decrease in droplet height against cycle number.

    (A) Real-time detection of 16S rRNA amplification during early cycles by DOTS qPCR at a thermocycling speed of 48 s per cycle. Percent decrease in droplet height is plotted against Cn for amplifications from 750, 75, 7.5, and 0.75 pg of genomic DNA (1.5 × 105, 1.5 × 104, 1.5 × 103, and 1.5 × 102 genomic copies, respectively) and NTC. Error bars represent overall device noise. A 4.8% threshold for detection is also plotted. The threshold was chosen to optimize the R2 value of the linear regression shown in Fig. 8. (B) Smartphone camera images of the DOT submerged in oil. Images were taken every 5 thermal cycles and used to determine the droplet height.

  • Fig. 6 Interfacial tension during DNA amplification in the presence of SG.

    Three reactions with different conditions were thermocycled in increments of 5 cycles. The reaction conditions were (i) 75 pg of K. pneumoniae genomic DNA (1.5 × 104 genomic copies) with SG to amplify the 16S rRNA V3 amplicon (196 bp), (ii) 75 pg of K. pneumoniae genomic DNA (1.5 × 104 genomic copies) without SG to amplify the 16S rRNA V3 amplicon (196 bp), and (iii) NTC with SG. The samples were analyzed by gel electrophoresis (fig. S1). (A) Band intensities at the 196-bp region of the gel images were quantified, normalized to the intensity at C0, and plotted against Cn. The product band is first detected at C20, and no product band is detected for the NTC. (B) Fluorescence qPCR amplification curve for the 16S rRNA V3 amplicon (196 bp) amplified from 75 pg of K. pneumoniae genomic DNA (1.5 × 104 genomic copies) and NTC. The Ct value is 21.11 ± 0.06. (C) The interfacial tensions of the PCRs were also analyzed with an FTÅ 200 contact angle and interfacial tension analyzer. The percent change in interfacial tension, dγ/γ0 = (γ0 − γn)/γ0, is plotted against Cn. The γ of the reaction with DNA and SG decreases by 21% by C10 and remains the same thereafter. The γ of the reaction with DNA but without SG increases by 11% by C5 and then further increases to 19% by C30. The γ of the SG NTC reaction increases by 6% by C5 and fluctuates within 4% thereafter.

  • Fig. 7 Femtoliter water-in-oil droplets after DNA amplification with SG.

    (A) Bright-field microscope image showing water-in-oil droplets stabilized by dsDNA/SG complexes. The droplets range from 1 to 2 μm in diameter and from 0.5 to 4.2 fl in volume. These femtoliter droplets are observed in the oil phase after DNA amplification with SG. (B) Molecular schematic illustrating adsorption at the oil-water interface: (a) protein adsorption initially stabilizes the droplet; (b) proteins undergo conformational change; (c) proteins form networks; (d) PCR produces dsDNA amplicons; (e) SG intercalates dsDNA, forming relatively hydrophobic complexes; (f and g) dsDNA/SG complexes replace the surface-bound proteins because of high interfacial affinity and high concentration (Vroman effect); (h) adsorption of dsDNA/SG complexes decreases interfacial tension, and colloidal suspensions become energetically favorable. Femtoliter water droplets are emulsified in the oil phase, decreasing DOT volume.

  • Fig. 8 Real-time PCR standard curves for DOTS qPCR and fluorescence qPCR.

    (A) DOTS qPCR standard curve for 16S amplification of the V3 amplicon from K. pneumoniae genomic DNA in the range of 1.5 × 102 to 1.5 × 105 genomic copies. A trend line is fitted to the data by linear regression analysis: log(N0) = −0.48Ct + 6.6; R2 = 0.981. In DOTS qPCR, the Ct values for NTC and 1.5 × 102, 1.5 × 103, 1.5 × 104, and 1.5 × 105 genomic copies are 14.4 ± 0.4, 9.0 ± 0.6, 7.5 ± 0.4, 4.6 ± 0.3, and 3.1 ± 0.2, respectively. (B) Fluorescence qPCR standard curve for 16S amplification of the V3 amplicon from K. pneumoniae genomic DNA in the same range. A trend line is fitted to the data by linear regression analysis: log(N0) = −0.24Ct + 9.4; R2 = 0.996. In fluorescence qPCR, the Ct values for NTC and 1.5 × 102, 1.5 × 103, 1.5 × 104, and 1.5 × 105 genomic copies are 32.4 ± 0.1, 29.88 ± 0.03, 25.28 ± 0.07, 21.11 ± 0.06, and 17.66 ± 0.04, respectively.

  • Table 1 Threshold cycles for DOTS qPCR and fluorescence qPCR.

    Uncertainties have been determined as the SE of repeated measurements for DOTS qPCR and as the SE of triplicate experiments for fluorescence qPCR.

    N0 (genomic copies)DOTS qPCR CtFluorescence qPCR Ct
    1.5 × 1053.1 ± 0.217.66 ± 0.04
    1.5 × 1044.6 ± 0.321.11 ± 0.06
    1.5 × 1037.5 ± 0.425.28 ± 0.07
    1.5 × 1029.0 ± 0.629.88 ± 0.03
    NTC14.4 ± 0.432.4 ± 0.1

Supplementary Materials

  • Supplementary material for this article is available at http://advances.sciencemag.org/cgi/content/full/1/8/e1400061/DC1

    Fig. S1. Gel electrophoresis of PCR amplification at different cycle numbers.

    Movie S1. DOTS qPCR device operation.

    Movie S2. Droplet imaging by smartphone.

    Movie S3. Close-up view of convective heating.

    Movie S4. Whole-device view of a complete thermal cycle.

  • Supplementary Materials

    This PDF file includes:

    • Fig. S1. Gel electrophoresis of PCR amplification at different cycle numbers.

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    Other Supplementary Material for this manuscript includes the following:

    • Movie S1 (.mp4 format). DOTS qPCR device operation.
    • Movie S2 (.mp4 format). Droplet imaging by smartphone.
    • Movie S3 (.mp4 format). Close-up view of convective heating.
    • Movie S4 (.mp4 format). Whole-device view of a complete thermal cycle.

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