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Issue 5, 2011
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An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents

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Abstract

We describe the first miniaturized device capable of the front-end sample preparation essential for detecting RNA-based infectious agents. The microfluidic device integrates sample purification and reverse transcription PCR (RT-PCR) amplification for the identification and detection of influenza A. The device incorporates a chitosan-based RNA binding phase for the completely aqueous isolation of nucleic acids, avoiding the PCR inhibitory effects of guanidine and isopropanol used in silica-based extraction methods. The purified nucleic acids and the reagents needed for single-step RT-PCR amplification are fluidically mobilized simultaneously to a PCR chamber. Utilizing infrared (IR)-mediated heating allowed for a > 5-fold decrease in RT-PCR analysis time compared to a standard thermal cycling protocol used in a conventional thermal cycler. Influenza A virus [A/PR/8/34 (H1N1)] was used as a simulant in this study for virus-based infectious and biowarfare agents with RNA genomes, and was successfully detected in a mock nasal swab sample at clinically relevant concentrations. Following on-chip purification, a fragment specific to the influenza A nucleoprotein gene was first amplified via RT-PCR amplification using IR-mediated heating to achieve more rapid heating and cooling rates. This was initially accomplished on a two-chip system to optimize the SPE and RT-PCR, and then translated to an integrated SPE-RT-PCR device.

Graphical abstract: An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents

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Publication details

The article was received on 21 Jun 2010, accepted on 22 Oct 2010 and first published on 08 Dec 2010


Article type: Technical Note
DOI: 10.1039/C0LC00136H
Citation: Lab Chip, 2011,11, 957-961
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    An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents

    K. A. Hagan, C. R. Reedy, M. L. Uchimoto, D. Basu, D. A. Engel and J. P. Landers, Lab Chip, 2011, 11, 957
    DOI: 10.1039/C0LC00136H

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