title: Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.
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.
authors.affiliation: Biomedical Diagnostics Institute, National Centre for Sensor Research, Research & Engineering Building, Dublin City University, Glasnevin, Dublin 9, Ireland.
authors.forename: Ivan K
authors.initials: IK
authors.lastname: Dimov
authors.forename: Jose L
authors.initials: JL
authors.lastname: Garcia-Cordero
authors.forename: Justin
authors.initials: J
authors.lastname: O'Grady
authors.forename: Claus R
authors.initials: CR
authors.lastname: Poulsen
authors.forename: Caroline
authors.initials: C
authors.lastname: Viguier
authors.forename: Lorcan
authors.initials: L
authors.lastname: Kent
authors.forename: Paul
authors.initials: P
authors.lastname: Daly
authors.forename: Bryan
authors.initials: B
authors.lastname: Lincoln
authors.forename: Majella
authors.initials: M
authors.lastname: Maher
authors.forename: Richard
authors.initials: R
authors.lastname: O'Kennedy
authors.forename: Terry J
authors.initials: TJ
authors.lastname: Smith
authors.forename: Antonio J
authors.initials: AJ
authors.lastname: Ricco
authors.forename: Luke P
authors.initials: LP
authors.lastname: Lee
mesh_terms: D019937:Diagnostic Techniques and Procedures; D004926:Escherichia coli / Q000737:chemistry; D044085:Microfluidics / Q000295:instrumentation / Q000379:methods; D012329:RNA, Bacterial / Q000737:chemistry*; D021161:Self-Sustained Sequence Replication / Q000295:instrumentation / Q000379:methods
chemical_list: D012329:RNA, Bacterial; C096222:tmRNA