Details

Autor(en) / Beteiligte

• Lagally, E. T
• Scherer, J. R
• Blazej, R. G
• Toriello, N. M
• Diep, B. A
• Ramchandani, M
• Sensabaugh, G. F
• Riley, L. W
• Mathies, R. A

Titel

Integrated Portable Genetic Analysis Microsystem for Pathogen/Infectious Disease Detection

Ist Teil von

• Analytical chemistry (Washington), 2004-06, Vol.76 (11), p.3162-3170

Ort / Verlag

Washington, DC: American Chemical Society

Erscheinungsjahr

2004

Quelle

MEDLINE

Beschreibungen/Notizen

• 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.