title: A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis.
abstract: Herein we present a fully automated system with pseudo-multiplexing capability for rapid infectious disease diagnosis. The all-in-one system was comprised of a polymer cartridge, a miniaturized thermal cycler, 1-color, 3-chamber fluorescence detectors for real-time reverse transcription polymerase chain reaction (RRT-PCR), and a pneumatic fluidic delivery unit consisting of two pinch-valve manifolds and two pneumatic pumps. The disposable, self-contained cartridge held all the necessary reagents for viral RNA purification and reverse transcription polymerase chain reaction (RT-PCR) detection, which took place all within the completely sealed cartridge. The operator only needed to pipette the patient's sample with lysis buffer into the cartridge, and the system would automatically perform the entire sample preparation and diagnosis within 2.5 h. We have successfully employed this system for seasonal influenza A H1N1 typing and sub-typing, obtaining comparable sensitivity as the experiments conducted using manual RNA extraction and commercial thermal cycler. A minimum detectable virus loading of 100 copies per μl has been determined by serial dilution experiments. This all-in-one desktop system would be suitable for decentralized disease diagnosis at immigration check points and outpatient clinics, and would not require highly skilled operators.
authors.affiliation: Institute of Bioengineering and Nanotechnology, The Nanos, Singapore.
authors.forename: Guolin
authors.initials: G
authors.lastname: Xu
authors.forename: Tseng-Ming
authors.initials: TM
authors.lastname: Hsieh
authors.forename: Daniel Y S
authors.initials: DY
authors.lastname: Lee
authors.forename: Emril Mohamed
authors.initials: EM
authors.lastname: Ali
authors.forename: Hong
authors.initials: H
authors.lastname: Xie
authors.forename: Xing Lun
authors.initials: XL
authors.lastname: Looi
authors.forename: Evelyn S-C
authors.initials: ES
authors.lastname: Koay
authors.forename: Mo-Huang
authors.initials: MH
authors.lastname: Li
authors.forename: Jackie Y
authors.initials: JY
authors.lastname: Ying
mesh_terms: D006801:Humans; D053118:Influenza A Virus, H1N1 Subtype / Q000145:classification / Q000235:genetics / Q000302:isolation & purification; D007251:Influenza, Human / Q000175:diagnosis* / Q000821:virology; D046210:Microfluidic Analytical Techniques / Q000295:instrumentation* / Q000379:methods; D009305:Nasopharynx / Q000821:virology; D019904:Polymethyl Methacrylate; D012367:RNA, Viral / Q000032:analysis / Q000302:isolation & purification; D011933:Reagent Kits, Diagnostic; D020133:Reverse Transcriptase Polymerase Chain Reaction / Q000295:instrumentation* / Q000379:methods; D013050:Spectrometry, Fluorescence; D013997:Time Factors
chemical_list: D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D019904:Polymethyl Methacrylate