Integrated microfluidic tmRNA purification and real-time NASBA device for molecular diagnostics.
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.
Dimov
Ivan K
IK
Biomedical Diagnostics Institute, National Centre for Sensor Research, Research & Engineering Building, Dublin City University, Glasnevin, Dublin 9, Ireland.
Garcia-Cordero
Jose L
JL
O'Grady
Justin
J
Poulsen
Claus R
CR
Viguier
Caroline
C
Kent
Lorcan
L
Daly
Paul
P
Lincoln
Bryan
B
Maher
Majella
M
O'Kennedy
Richard
R
Smith
Terry J
TJ
Ricco
Antonio J
AJ
Lee
Luke P
LP
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
D012329:RNA, Bacterial; C096222:tmRNA