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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

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%.
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Figure 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%.

Mentions: We designed the DOTS qPCR device (Fig. 1, A and B) to be readily deployed as a point-of-care diagnostic tool and to epitomize simplicity, small form factor, mobile integration, and disposability. The DOT (Fig. 1C) is submerged in a heated oil environment and is positioned by a motor (Fig. 1, D to F, and movies S1 to S4). The oil is contained within a semicircular channel with two heaters, located at 0° and 180°, which maintain the two temperature extremes (45° to 50°C and 100° to 105°C). A heat gradient is established along the channel with temperatures between the two extremes being represented (Fig. 2A). From room temperature (25°C), the steady state of the heat gradient is established within 10 min of commencing temperature ramping using proportional-integral-derivative (PID) control of the heater power (Fig. 2B). At the midpoint of the channel, a viewing window allows macroscopic imaging of the droplet by a smartphone camera with an attached lens. The oil temperature at this window is 70°C. The internal temperature of the droplet is continuously monitored by a thermocouple, which is bent such that the thermocouple junction is positioned inside the droplet (Fig. 1C). The position of the droplet within the heat gradient is accurately controlled using real-time feedback of its internal temperature.


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)

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%.
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
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Figure 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%.
Mentions: We designed the DOTS qPCR device (Fig. 1, A and B) to be readily deployed as a point-of-care diagnostic tool and to epitomize simplicity, small form factor, mobile integration, and disposability. The DOT (Fig. 1C) is submerged in a heated oil environment and is positioned by a motor (Fig. 1, D to F, and movies S1 to S4). The oil is contained within a semicircular channel with two heaters, located at 0° and 180°, which maintain the two temperature extremes (45° to 50°C and 100° to 105°C). A heat gradient is established along the channel with temperatures between the two extremes being represented (Fig. 2A). From room temperature (25°C), the steady state of the heat gradient is established within 10 min of commencing temperature ramping using proportional-integral-derivative (PID) control of the heater power (Fig. 2B). At the midpoint of the channel, a viewing window allows macroscopic imaging of the droplet by a smartphone camera with an attached lens. The oil temperature at this window is 70°C. The internal temperature of the droplet is continuously monitored by a thermocouple, which is bent such that the thermocouple junction is positioned inside the droplet (Fig. 1C). The position of the droplet within the heat gradient is accurately controlled using real-time feedback of its internal temperature.

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