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

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Authors

  • 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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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.

Details

Original languageEnglish
Pages (from-to)2071-2078
Number of pages8
JournalLab on a Chip
Volume8
Issue number12
DOIs
Publication statusPublished - 2008
Peer-reviewedYes

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