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

Dimov, Ivan K, Garcia-Cordero, Jose L, O'Grady, Justin, Poulsen, Claus R, Viguier, Caroline, Kent, Lorcan, Daly, Paul, Lincoln, Bryan, Maher, Majella, O'Kennedy, Richard, Smith, Terry J, Ricco, Antonio J and Lee, Luke P (2008) Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics. Lab on a Chip, 8 (12). pp. 2071-2078. ISSN 1473-0197

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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.

Item Type: Article
Faculty \ School: Faculty of Medicine and Health Sciences > Norwich Medical School
Related URLs:
Depositing User: Justin O'Grady
Date Deposited: 10 Sep 2013 04:59
Last Modified: 21 Apr 2020 17:59
URI: https://ueaeprints.uea.ac.uk/id/eprint/41317
DOI: 10.1039/b812515e

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