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Mandatory Fields
Dimov, IK,Garcia-Cordero, JL,O'Grady, J,Poulsen, CR,Viguier, C,Kent, L,Daly, P,Lincoln, B,Maher, M,O'Kennedy, R,Smith, TJ,Ricco, AJ,Lee, LP
2008
July
Lab On A Chip
Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics
Published
()
Optional Fields
SEQUENCE-BASED AMPLIFICATION DNA EXTRACTION LISTERIA-MONOCYTOGENES SAMPLE PREPARATION PCR DETECTION CELL-LYSIS BEACONS SYSTEMS IDENTIFICATION PATHOGENS
8
2071
2078
We demonstrate the first integrated microfluidic tmRNA purification and nucleic acid sequence-based amplification (NASBA) device incorporating real-time detection. The real-time amplification and detection step produces pathogen-specific response in < 3 min from the chip-purified RNA from 100 lysed bacteria. On-chip RNA purification uses a new silica bead immobilization method. On-chip amplification uses custom-designed high-selectivity primers and real-time detection uses molecular beacon fluorescent probe technology; both are integrated on-chip with NASBA. Present in all bacteria, tmRNA (10Sa RNA) includes organism-specific identification sequences, exhibits unusually high stability relative to mRNA, and has high copy number per organism; the latter two factors improve the limit of detection, accelerate time-to-positive response, and suit this approach ideally to the detection of small numbers of bacteria. Device efficacy was demonstrated by integrated on-chip purification, amplification, and real-time detection of 100 E. coli bacteria in 100 mu L of crude lysate in under 30 min for the entire process.
DOI 10.1039/b812515e
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