Journal
BIOMEDICAL MICRODEVICES
Volume 15, Issue 2, Pages 221-231Publisher
SPRINGER
DOI: 10.1007/s10544-012-9720-1
Keywords
Microfluidics; Sensitivity; Virus detection; Polymerase chain reaction; Consensus degenerate; Passivation
Funding
- NSF [CISE 1110947, EHR 0965945]
- American Heart Association [10GRNT4430029]
- Georgia Economic Development Association
- Wallace H. Coulter Foundation
- Centers for Disease Control and Prevention (CDC)
- NSF National Nanotechnology Infrastructure Network (NNIN)
- Georgia Tech: Institute for Bioengineering and Biosciences Junior Faculty Award
- Technology Fee Fund
- Invention Studio
- George W. Woodruff School of Mechanical Engineering
- Department of Homeland Security (DHS) Scholarship
- Fellowship Program
- DOE [DE-AC05-06OR23100]
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Sensitive identification of the etiology of viral diseases is key to implementing appropriate prevention and treatment. The gold standard for virus identification is the polymerase chain reaction (PCR), a technique that allows for highly specific and sensitive detection of pathogens by exponentially amplifying a specific region of DNA from as little as a single copy through thermocycling a biochemical cocktail. Today, molecular biology laboratories use commercial instruments that operate in 0.5-2 h/analysis using reaction volumes of 5-50 mu L contained within polymer tubes or chambers. Towards reducing this volume and maintaining performance, we present a semi-quantitative, systematic experimental study of how PCR yield is affected by tube/chamber substrate, surface-area-to-volume ratio (SA:V), and passivation methods. We perform PCR experiments using traditional PCR tubes as well as using disposable polymer microchips with 1 mu L reaction volumes thermocycled using water baths. We report the first oil encapsulation microfluidic PCR method without fluid flow and its application to the first microfluidic amplification of Epstein Barr virus using consensus degenerate primers, a powerful and broad PCR method to screen for both known and novel members of a viral family. The limit of detection is measured as 140 starting copies of DNA from a starting concentration of 3 x 10(5) copies/mL, regarded as an accepted sensitivity threshold for diagnostic purposes, and reaction specificity was improved as compared to conventional methods. Also notable, these experiments were conducted with conventional reagent concentrations, rather than commonly spiked enzyme and/or template mixtures. This experimental study of the effects of substrate, SA:V, and passivation, together with sensitive and specific microfluidic PCR with consensus degenerate primers represent advances towards lower cost and higher throughput pathogen screening.
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