Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.

Ivan K Dimov, Jose L Garcia-Cordero, Justin O'Grady, Claus R Poulsen, Caroline Viguier, Lorcan Kent, Paul Daly, Bryan Lincoln, Majella Maher, Richard O'Kennedy, Terry J Smith, Antonio J Ricco, Luke P Lee

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114 Citations (Scopus)
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Abstract

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 microL of crude lysate in under 30 min for the entire process.
Original languageEnglish
Pages (from-to)2071-2078
Number of pages8
JournalLab on a Chip
Volume8
Issue number12
DOIs
Publication statusPublished - 2008

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