tetano
Editor, Senior Moderator
Lab Chip. 2010 Dec 8. [Epub ahead of print]
An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.
Hagan KA, Reedy CR, Uchimoto ML, Basu D, Engel DA, Landers JP.
Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA 22904, USA. landers@virginia.edu.
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.
PMID: 21152489 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21152489
An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.
Hagan KA, Reedy CR, Uchimoto ML, Basu D, Engel DA, Landers JP.
Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA 22904, USA. landers@virginia.edu.
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.
PMID: 21152489 [PubMed - as supplied by publisher]
http://www.ncbi.nlm.nih.gov/pubmed/21152489