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Issue 12, 2008
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Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

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

Graphical abstract: Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics

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Submitted
21 Jul 2008
Accepted
17 Sep 2008
First published
24 Oct 2008

Lab Chip, 2008,8, 2071-2078
Article type
Paper

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

I. K. Dimov, J. L. Garcia-Cordero, J. O'Grady, C. R. Poulsen, C. Viguier, L. Kent, P. Daly, B. Lincoln, M. Maher, R. O'Kennedy, T. J. Smith, A. J. Ricco and L. P. Lee, Lab Chip, 2008, 8, 2071
DOI: 10.1039/B812515E

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