A self-contained all-in-one cartridge for sample preparation and real-time PCR in rapid influenza diagnosis.
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.
Xu
Guolin
G
Institute of Bioengineering and Nanotechnology, The Nanos, Singapore.
Hsieh
Tseng-Ming
TM
Lee
Daniel Y S
DY
Ali
Emril Mohamed
EM
Xie
Hong
H
Looi
Xing Lun
XL
Koay
Evelyn S-C
ES
Li
Mo-Huang
MH
Ying
Jackie Y
JY
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
D012367:RNA, Viral; D011933:Reagent Kits, Diagnostic; D019904:Polymethyl Methacrylate