title: An integrated, valveless system for microfluidic purification and reverse transcription-PCR amplification of RNA for detection of infectious agents.
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.
authors.affiliation: Department of Chemistry, University of Virginia, McCormick Road, P.O. Box 400319, Charlottesville, VA 22904, USA.
authors.forename: Kristin A
authors.initials: KA
authors.lastname: Hagan
authors.forename: Carmen R
authors.initials: CR
authors.lastname: Reedy
authors.forename: Mari L
authors.initials: ML
authors.lastname: Uchimoto
authors.forename: Dipanwita
authors.initials: D
authors.lastname: Basu
authors.forename: Daniel A
authors.initials: DA
authors.lastname: Engel
authors.forename: James P
authors.initials: JP
authors.lastname: Landers
mesh_terms: D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000235:genetics / Q000302:isolation & purification*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D009666:Nose / Q000821:virology; D012367:RNA, Viral / Q000032:analysis / Q000235:genetics* / Q000302:isolation & purification*; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation*; D052616:Solid Phase Extraction; D018511:Systems Integration; D013997:Time Factors
chemical_list: D012367:RNA, Viral