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

Dimov, Ivan K and Garcia-Cordero, Jose L and O'Grady, Justin and Poulsen, Claus R and Viguier, Caroline and Kent, Lorcan and Daly, Paul and Lincoln, Bryan and Maher, Majella and O'Kennedy, Richard and Smith, Terry J and 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. 2071-8. ISSN 1473-0197 (Print); 1473-0189 (Electronic)

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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
Subjects:Biomedical Science > Nanomedicine
ID Code:7785
Deposited By:M T V
Deposited On:23 Nov 2009 13:30
Last Modified:23 Nov 2009 13:30

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