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Micromachines 2015, 6(1), 63-79; doi:10.3390/mi6010063

Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology

1
Biosystems Research and Applications Group, Department of Electrical and Computer Engineering, Schulich School of Engineering, University of Calgary, Calgary, AB T2N 1N4, Canada
2
Provincial Laboratory for Public Health of Alberta, Calgary, AB T2N 4W4, Canada
3
Department of Microbiology, Immunology and Infectious Diseases, Cumming School of Medicine, University of Calgary, Calgary, AB T2N 1N4, Canada
*
Author to whom correspondence should be addressed.
Academic Editor: Joost Lötters
Received: 18 November 2014 / Accepted: 16 December 2014 / Published: 23 December 2014
(This article belongs to the Special Issue Biomedical Microdevices)
View Full-Text   |   Download PDF [7608 KB, uploaded 29 December 2014]   |  

Abstract

Quantitative, reverse transcription, polymerase chain reaction (qRT-PCR) is facilitated by leveraging droplet microfluidic (DMF) system, which due to its precision dispensing and sample handling capabilities at microliter and lower volumes has emerged as a popular method for miniaturization of the PCR platform. This work substantially improves and extends the functional capabilities of our previously demonstrated single qRT-PCR micro-chip, which utilized a combination of electrostatic and electrowetting droplet actuation. In the reported work we illustrate a spatially multiplexed micro-device that is capable of conducting up to eight parallel, real-time PCR reactions per usage, with adjustable control on the PCR thermal cycling parameters (both process time and temperature set-points). This micro-device has been utilized to detect and quantify the presence of two clinically relevant respiratory viruses, Influenza A and Influenza B, in human samples (nasopharyngeal swabs, throat swabs). The device performed accurate detection and quantification of the two respiratory viruses, over several orders of RNA copy counts, in unknown (blind) panels of extracted patient samples with acceptably high PCR efficiency (>94%). The multi-stage qRT-PCR assays on eight panel patient samples were accomplished within 35–40 min, with a detection limit for the target Influenza virus RNAs estimated to be less than 10 RNA copies per reaction. View Full-Text
Keywords: reverse transcription PCR (RT-PCR); Influenza viruses; droplet microfluidics (DMF); nano-texture; dielectrophoresis (DEP); electrostatics; electrowetting (EW); multiplex qRT-PCR assays reverse transcription PCR (RT-PCR); Influenza viruses; droplet microfluidics (DMF); nano-texture; dielectrophoresis (DEP); electrostatics; electrowetting (EW); multiplex qRT-PCR assays
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This is an open access article distributed under the Creative Commons Attribution License which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited. (CC BY 4.0).

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MDPI and ACS Style

Prakash, R.; Pabbaraju, K.; Wong, S.; Wong, A.; Tellier, R.; Kaler, K.V.I.S. Multiplex, Quantitative, Reverse Transcription PCR Detection of Influenza Viruses Using Droplet Microfluidic Technology. Micromachines 2015, 6, 63-79.

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