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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

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.
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Figure 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.

Mentions: (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.


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)

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.
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Related In: Results  -  Collection

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Figure 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.
Mentions: (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.

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