title: Integrated portable genetic analysis microsystem for pathogen/infectious disease detection.
abstract: An integrated portable genetic analysis microsystem including PCR amplification and capillary electrophoretic (CE) analysis coupled with a compact instrument for electrical control and laser-excited fluorescence detection has been developed. The microdevice contains microfabricated heaters, temperature sensors, and membrane valves to provide controlled sample positioning and immobilization in 200-nL PCR chambers. The instrument incorporates a solid-state laser and confocal fluorescence detection optics, electronics for sensing and powering the PCR reactor, and high-voltage power supplies for conducting CE separations. The fluorescein-labeled PCR products are amplified and electrophoretically analyzed in a gel-filled microchannel in <10 min. We demonstrate the utility of this instrument by performing pathogen detection and genotyping directly from whole Escherichia coli and Staphylococcus aureus cells. The E. coli detection assay consists of a triplex PCR amplification targeting genes that encode 16S ribosomal RNA, the fliC flagellar antigen, and the sltI shigatoxin. Serial dilution demonstrates a limit of detection of 2-3 bacterial cells. The S. aureus assay uses a femA marker to identify cells as S. aureus and a mecA marker to probe for methicillin resistance. This integrated portable genomic analysis microsystem demonstrates the feasibility of performing rapid high-quality detection of pathogens and their antimicrobial drug resistance.
authors.affiliation: Chemistry Department, University of California, Berkeley, California 94720, USA.
authors.forename: E T
authors.initials: ET
authors.lastname: Lagally
authors.forename: J R
authors.initials: JR
authors.lastname: Scherer
authors.forename: R G
authors.initials: RG
authors.lastname: Blazej
authors.forename: N M
authors.initials: NM
authors.lastname: Toriello
authors.forename: B A
authors.initials: BA
authors.lastname: Diep
authors.forename: M
authors.initials: M
authors.lastname: Ramchandani
authors.forename: G F
authors.initials: GF
authors.lastname: Sensabaugh
authors.forename: L W
authors.initials: LW
authors.lastname: Riley
authors.forename: R A
authors.initials: RA
authors.lastname: Mathies
mesh_terms: D000818:Animals; D001419:Bacteria / Q000235:genetics / Q000302:isolation & purification*; D001424:Bacterial Infections / Q000175:diagnosis*; D001483:Base Sequence; D019075:Electrophoresis, Capillary / Q000379:methods*; D004927:Escherichia coli Infections / Q000175:diagnosis; D008969:Molecular Sequence Data; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D016133:Polymerase Chain Reaction / Q000379:methods*; D013203:Staphylococcal Infections / Q000175:diagnosis