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

doi:10.1039/B812515E

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Lab on a Chip

Issue 12, 2008

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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 and Luke P. Lee

Lab Chip, 2008, 8, 2071-2078

DOI: 10.1039/B812515E
Received 21 Jul 2008, Accepted 17 Sep 2008
First published on the web 24 Oct 2008
This article is part of the collection: Microfluidics and point-of-care testing

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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 µL of crude lysate in under 30 min for the entire process.

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