Integrated portable genetic analysis microsystem for pathogen/infectious disease detection

Anal Chem. 2004 Jun 1;76(11):3162-70. doi: 10.1021/ac035310p.

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.

Publication types

  • Research Support, Non-U.S. Gov't
  • Research Support, U.S. Gov't, P.H.S.

MeSH terms

  • Animals
  • Bacteria / genetics
  • Bacteria / isolation & purification*
  • Bacterial Infections / diagnosis*
  • Base Sequence
  • Electrophoresis, Capillary / methods*
  • Escherichia coli Infections / diagnosis
  • Molecular Sequence Data
  • Oligonucleotide Array Sequence Analysis / methods*
  • Polymerase Chain Reaction / methods*
  • Staphylococcal Infections / diagnosis