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

Dustin K. Harshman, Brianna M. Rao, Jean E T Mclain, George S Watts, Jeong-Yeol Yoon

Research output: Contribution to journalArticle

8 Citations (Scopus)

Abstract

Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decisionmaking. 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 sampleto- 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.

Original languageEnglish (US)
Article numbere1400061
JournalScience advances
Volume1
Issue number8
DOIs
StatePublished - Sep 1 2015

Fingerprint

Real-Time Polymerase Chain Reaction
Point-of-Care Systems
Optical Devices
Access to Information
Surface Tension
Molecular Pathology
Computer Systems
Endocarditis
Cell Nucleus
Adsorption
Limit of Detection
Fluorescence
Anti-Bacterial Agents
Technology
DNA
Infection
Research
Smartphone

ASJC Scopus subject areas

  • Medicine(all)

Cite this

Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief. / Harshman, Dustin K.; Rao, Brianna M.; Mclain, Jean E T; Watts, George S; Yoon, Jeong-Yeol.

In: Science advances, Vol. 1, No. 8, e1400061, 01.09.2015.

Research output: Contribution to journalArticle

@article{726b9ee8d98341d5bdbce2e284166be8,
title = "Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief",
abstract = "Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decisionmaking. 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 sampleto- 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.",
author = "Harshman, {Dustin K.} and Rao, {Brianna M.} and Mclain, {Jean E T} and Watts, {George S} and Jeong-Yeol Yoon",
year = "2015",
month = "9",
day = "1",
doi = "10.1126/sciadv.1400061",
language = "English (US)",
volume = "1",
journal = "Science advances",
issn = "2375-2548",
publisher = "American Association for the Advancement of Science",
number = "8",

}

TY - JOUR

T1 - Innovative qPCR using interfacial effects to enable low threshold cycle detection and inhibition relief

AU - Harshman, Dustin K.

AU - Rao, Brianna M.

AU - Mclain, Jean E T

AU - Watts, George S

AU - Yoon, Jeong-Yeol

PY - 2015/9/1

Y1 - 2015/9/1

N2 - Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decisionmaking. 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 sampleto- 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.

AB - Molecular diagnostics offers quick access to information but fails to operate at a speed required for clinical decisionmaking. 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 sampleto- 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.

UR - http://www.scopus.com/inward/record.url?scp=85032850168&partnerID=8YFLogxK

UR - http://www.scopus.com/inward/citedby.url?scp=85032850168&partnerID=8YFLogxK

U2 - 10.1126/sciadv.1400061

DO - 10.1126/sciadv.1400061

M3 - Article

AN - SCOPUS:85032850168

VL - 1

JO - Science advances

JF - Science advances

SN - 2375-2548

IS - 8

M1 - e1400061

ER -