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

Harshman DK, Rao BM, McLain JE, Watts GS, Yoon JY - Sci Adv (2015)

Bottom Line: Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone.Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses.Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Engineering Graduate Interdisciplinary Program, The University of Arizona, Tucson, AZ 85721, USA.

ABSTRACT
Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

No MeSH data available.


Related in: MedlinePlus

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.
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Figure 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.

Mentions: A porcine model for IE was developed (33) (Fig. 3F). Excised heart valve tissue punches (6-mm-diameter sections) (Fig. 3G) were sterilized, inoculated with vancomycin-resistant Enterococcus faecium (VRE), and ground using a micro–mortar and pestle. The liquid phase of the tissue after grinding had a protein concentration of 1.6 ± 0.1 mg/ml (Fig. 3A). The interfacial tensions (γ) of the PCR cocktail with the purified target and the PCR cocktail with the tissue-contaminated target were measured with a First Ten Ångstroms (FTÅ) 200 contact angle and interfacial tension analyzer, and the interfacial tensions were 25.55 and 27.60 mN/m, respectively (Fig. 3B). A free-body force diagram illustrates the direction of the forces acting on the DOT (Fig. 3C). Because of the interfacial tension force Fγ, a droplet of the PCR mixture can be suspended on the thermocouple loop. In fluorescence qPCR, tissue proteins inhibit amplification of the 16S rRNA gene V1-V2 hypervariable regions and the antibiotic resistance gene vanA from intact VRE. Therefore, the threshold cycles (Ct) are shifted upward by 1.6 cycles for the 16S rRNA V1-V2 reaction and by 5.4 cycles for the vanA reaction (Fig. 3D). In DOT thermocycling, these tissue proteins should be adsorbed at the oil-water interface, so that contaminating proteins are effectively eliminated from the PCR (interfacial compartmentalization). However, Taq polymerase should not be adsorbed at the oil-water interface. To explain this, the diffusion amounts of the relevant blood and tissue proteins (34) to the oil-water interface were calculated for comparison with the diffusion of Taq polymerase (Fig. 3E). The following proteins were included in the calculation, with the corresponding molecular weights and diffusivities: albumin (94 kD, 6.1 × 10−7 cm2/s), immunoglobulin G (150 kD, 4.0 × 10−7 cm2/s), fibrinogen (340 kD, 2.0 × 10−7 cm2/s) (35), fibronectin (450 kD, 0.9 × 10−7 cm2/s) (36), collagen type I (282 kD, 0.78 × 10−7 cm2/s) (37), tropoelastin (65 kD, 4.6 × 10−7 cm2/s) (38), and Taq polymerase (94 kD, 4.7 × 10−7 cm2/s) (39). As shown in Fig. 3G, the amounts of albumin and fibrinogen absorbed are several orders of magnitude greater than that of Taq polymerase.


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

Harshman DK, Rao BM, McLain JE, Watts GS, Yoon JY - Sci Adv (2015)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: A porcine model for IE was developed (33) (Fig. 3F). Excised heart valve tissue punches (6-mm-diameter sections) (Fig. 3G) were sterilized, inoculated with vancomycin-resistant Enterococcus faecium (VRE), and ground using a micro–mortar and pestle. The liquid phase of the tissue after grinding had a protein concentration of 1.6 ± 0.1 mg/ml (Fig. 3A). The interfacial tensions (γ) of the PCR cocktail with the purified target and the PCR cocktail with the tissue-contaminated target were measured with a First Ten Ångstroms (FTÅ) 200 contact angle and interfacial tension analyzer, and the interfacial tensions were 25.55 and 27.60 mN/m, respectively (Fig. 3B). A free-body force diagram illustrates the direction of the forces acting on the DOT (Fig. 3C). Because of the interfacial tension force Fγ, a droplet of the PCR mixture can be suspended on the thermocouple loop. In fluorescence qPCR, tissue proteins inhibit amplification of the 16S rRNA gene V1-V2 hypervariable regions and the antibiotic resistance gene vanA from intact VRE. Therefore, the threshold cycles (Ct) are shifted upward by 1.6 cycles for the 16S rRNA V1-V2 reaction and by 5.4 cycles for the vanA reaction (Fig. 3D). In DOT thermocycling, these tissue proteins should be adsorbed at the oil-water interface, so that contaminating proteins are effectively eliminated from the PCR (interfacial compartmentalization). However, Taq polymerase should not be adsorbed at the oil-water interface. To explain this, the diffusion amounts of the relevant blood and tissue proteins (34) to the oil-water interface were calculated for comparison with the diffusion of Taq polymerase (Fig. 3E). The following proteins were included in the calculation, with the corresponding molecular weights and diffusivities: albumin (94 kD, 6.1 × 10−7 cm2/s), immunoglobulin G (150 kD, 4.0 × 10−7 cm2/s), fibrinogen (340 kD, 2.0 × 10−7 cm2/s) (35), fibronectin (450 kD, 0.9 × 10−7 cm2/s) (36), collagen type I (282 kD, 0.78 × 10−7 cm2/s) (37), tropoelastin (65 kD, 4.6 × 10−7 cm2/s) (38), and Taq polymerase (94 kD, 4.7 × 10−7 cm2/s) (39). As shown in Fig. 3G, the amounts of albumin and fibrinogen absorbed are several orders of magnitude greater than that of Taq polymerase.

Bottom Line: Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone.Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses.Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

View Article: PubMed Central - PubMed

Affiliation: Biomedical Engineering Graduate Interdisciplinary Program, The University of Arizona, Tucson, AZ 85721, USA.

ABSTRACT
Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decision-making. Our novel methodology, droplet-on-thermocouple silhouette real-time polymerase chain reaction (DOTS qPCR), uses interfacial effects for droplet actuation, inhibition relief, and amplification sensing. DOTS qPCR has sample-to-answer times as short as 3 min 30 s. In infective endocarditis diagnosis, DOTS qPCR demonstrates reproducibility, differentiation of antibiotic susceptibility, subpicogram limit of detection, and thermocycling speeds of up to 28 s/cycle in the presence of tissue contaminants. Langmuir and Gibbs adsorption isotherms are used to describe the decreasing interfacial tension upon amplification. Moreover, a log-linear relationship with low threshold cycles is presented for real-time quantification by imaging the droplet-on-thermocouple silhouette with a smartphone. DOTS qPCR resolves several limitations of commercially available real-time PCR systems, which rely on fluorescence detection, have substantially higher threshold cycles, and require expensive optical components and extensive sample preparation. Due to the advantages of low threshold cycle detection, we anticipate extending this technology to biological research applications such as single cell, single nucleus, and single DNA molecule analyses. Our work is the first demonstrated use of interfacial effects for sensing reaction progress, and it will enable point-of-care molecular diagnosis of infections.

No MeSH data available.


Related in: MedlinePlus