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Abstract
Bird flu or Avian flu is an infectious disease caused by an influenza A virus of the
Orthomyxoviridae family. Avian influenza virus (AIV) causes significant economic losses to the
poultry industry worldwide and threatens human life with a pandemic. Pandemic of AIV is the
human infection caused by the appearance of a “new” influenza virus as a result of antigenic
shift or antigenic drift. Several outbreaks of AIV caused by the rapid spread of infection have
been identified. Therefore, there is an urgent need for rapid diagnostic methods that would
enable early detection and improve measurements to control the AIV outbreak. Classical method
for detection and identification of AIV is time consuming (3-10 days), laborious, less sensitive,
and requires special laboratory facilities and trained staff. Molecular diagnostic systems using
RT-PCR amplification have significantly improved the speed, sensitivity and specificity of
detecting AIV but are still cumbersome, expensive and time-consuming (1-2 days). In both
classical and molecular diagnosis, the transportation of sample to the near-by reference or
diagnostic laboratory is needed, and this will increases the time for diagnostic result. A simple
approach would be to have a point-of-care (POC) diagnostic test at or near the site of sample
collection to provide results in very short time and can improve medical decision-making. The
available commercial POC tests that are used for screening of influenza A virus are rapid (5-30
minutes) but have low sensitivity and false negative results are of major concern. Ultimately, the
miniaturization of the molecular diagnostics using Lab-on-a-chip (LOC) systems could provide
the next-generation rapid POC diagnostics.
This study has been focused on developing rapid diagnostic methods for the identification and
subtyping of the AIV towards POC diagnosis. The first step in molecular diagnostic is sample
preparation which is the key to the success of diagnosis. To address this, a novel method was
developed for selective separation and purification of AIV from chicken faecal sample using
monoclonal antibody (mAb) conjugated magnetic beads where RNA extraction step is not
required. The developed bead-based system was able to capture, concentrate and purify all of the
16 H subtypes of AIV from the AIV spiked faecal samples, demonstrating the efficiency of the
mAb conjugated beads and the developed method. Subsequently, the newly developed beadiv
based method was used in a microfluidic magnetic microsystem for the automation of sample
preparation. Using LOC system with a Cyclic-Olefin-Copolymer (COC) polymer chip, the RTPCR
was miniaturized and the entire process was detected in less than 2 h. This integrated LOC
system for has a great potential for POC clinical diagnostics.
Subtyping of AIV is important in the diagnosis to identify the pathogenic virus. A DNA
microarray-based solid-phase PCR approach has been developed for rapid detection of influenza
virus types A and simultaneous identification of pathogenic virus subtypes of H5 and H7. This
solid-phase RT-PCR method combines a reverse-transcription amplification of RNA extract in
the liquid-phase with sequence-specific nested PCR on the solid phase. The examination of 33
avian faecal and tracheal swab specimens was completed in less than 2 h with 94% accuracy.
Subsequently, the approach of solid-phase-PCR was extended to a microfluidic chip to reduce
sample and reagent consumption. The whole processing time for identifying and simultaneously
subtyping AIV was further reduced to 1h.
Apart from the RT-PCR method, two immunological methods based on; fluorescent DNA
barcode and fluorescent beads were also developed for rapid detection and identification of the
AIV. In both methods, the detection involved sandwiching of the target AIV between
monoclonal antibodies for nucleoproteins and for matrix proteins. In the fluorescent DNA
barcode-based immunoassay, fluorophore-tagged oligonucleotides were used as surrogates for
signal detection with sensitivity comparable to conventional RT-PCR for allantoic fluid
containing H16N3 AIV. While in the fluorescent bead-based immunoassay, the fluorescent
beads were used as the direct detection signal from AIV. In both methods the entire detection
time was less than 2 h.
Original language | English |
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Place of Publication | Kgs. Lyngby, Denmark |
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Publisher | Technical University of Denmark |
Number of pages | 139 |
Publication status | Published - Mar 2011 |
Keywords
- RT-PCR
- Solidphase PCR
- Magnetic Beads
- Magnetic microsystem
- Biobarcode immunoassay
- Faecal sample
- Sample Preparation
- Microfluidics
- Microarray
- Lab-on-a-chip
- Diagnostics
- Fluorescent beads
- Avian Influenza Virus
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Dive into the research topics of 'Rapid detection of Avian Influenza Virus - Towards point of care diagnosis'. Together they form a unique fingerprint.Projects
- 1 Finished
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Microarray for detecting avian influenza (Aiv) suitable for Lab-on-a-Chip applications
Dhumpa, R. (PhD Student), Bang, D. D. (Main Supervisor), Wolff, A. (Supervisor), Emnéus, J. (Examiner), Joos, T. O. (Examiner) & Nielsen, L. P. (Examiner)
01/01/2008 → 13/04/2011
Project: PhD