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

TitleInnovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief.
Publication TypeJournal Article
Year of Publication2015
AuthorsHarshman DK, Rao BM, McLain JE, Watts GS, Yoon J-Y
JournalSci Adv
Volume1
Issue8
Paginatione1400061
Date Published2015 Sep
ISSN2375-2548
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.

DOI10.1126/sciadv.1400061
Alternate JournalSci Adv
PubMed ID26601245
PubMed Central IDPMC4643774
Grant ListP30 CA023074 / CA / NCI NIH HHS / United States
P30 ES006694 / ES / NIEHS NIH HHS / United States
T32 HL007955 / HL / NHLBI NIH HHS / United States
Faculty Member Reference: 
George Watts, Ph.D.