Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief
AffiliationBiomedical Engineering Graduate Interdisciplinary Program, The University of Arizona
Department of Biomedical Engineering, The University of Arizona
Water Resources Research Center and Department of Soil, Water and Environmental Science, The University of Arizona
Arizona Cancer Center and Department of Pharmacology, The University of Arizona
Department of Agricultural and Biosystems Engineering, The University of Arizona
rapid molecular diagnostics
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CitationInnovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief 2015, 1 (8):e1400061 Science Advances
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AbstractMolecular 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.
DescriptionUA Open Access Publishing Fund
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