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Development of a magnetic bead microarray for simultaneous and simple detection of four pestiviruses
SLU , Uppsala.
KTH, School of Biotechnology (BIO), Nano Biotechnology. (Nanobiotechnology)
KTH, School of Biotechnology (BIO), Nano Biotechnology.ORCID iD: 0000-0001-8141-8449
SLU, Uppsala.
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2009 (English)In: Journal of Virological Methods, ISSN 0166-0934, Vol. 155, 1-9 p.Article in journal (Refereed) Published
Abstract [en]

This study reports a novel method for the rapid detection and identification of the four recognized species in the pestivirus genus of the Flaviviridae family, i.e. classical swine fever virus (CSFV), border disease virus (BDV), bovine viral diarrhoea virus type 1 (BVDV1) and type 2 (BVDV2). The analysis of pestivirus PCR products was performed on microarrays by means of magnetic bead detection. The process utilizes an oligonucleotide array, onto which 5' biotinylated PCR products were hybridized, followed by visualization with streptavidin-coated magnetic particles by the naked eye, microscope or biochip reader. The assay was tested on a collection of pestiviruses that included all four species and allowed a specific and sensitive detection. Sensitivity was compared with other post-PCR detection methods, namely gel electrophoresis and suspension microarray. The results indicate that due to its high sensitivity, specificity and simple detection procedure, the magnetic bead assay provides a powerful tool for detection and identification of viral pathogens. Considering the simplicity of the assay, the protocols for hybridization and magnetic bead detection offer an emerging application for molecular diagnoses in virology that is amenable for use in a modestly equipped laboratory.

Place, publisher, year, edition, pages
2009. Vol. 155, 1-9 p.
Keyword [en]
Pestivirus; CSFV; BDV; BVDV1; BVDV2; PCR; Magnetic bead; Microarray
National Category
Industrial Biotechnology
URN: urn:nbn:se:kth:diva-11219DOI: 10.1016/j.jviromet.2008.04.010ISI: 000262573400001ScopusID: 2-s2.0-57549108180OAI: diva2:242211
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-01-20Bibliographically approved
In thesis
1. Novel diagnostic microarray assay formats towards comprehensive on-site analysis
Open this publication in new window or tab >>Novel diagnostic microarray assay formats towards comprehensive on-site analysis
2009 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Advances in molecular methods for analyzing DNA, RNA and proteins in humans as well as in other animals, plants, fungi, bacteria or viruses have greatly increased the resolution with which we can study life’s complexity and dynamics on earth. While genomic, transcriptomic and proteomic laboratory tools for molecular diagnosis of disease are rapidly becoming more comprehensive, the access to such advanced yet often expensive and centralized procedures is limited. There is a great need for rapid and comprehensive diagnostic methods in low-resource settings or contexts where a person can not or will not go to a hospital or medical laboratory, yet where a clinical analysis is urgent.

In this thesis, results from development and characterization of novel technologies for DNA and protein microarray analysis are presented. Emphasis is on methods that could provide rapid, cost-effective and portable analysis with convenient readout and retained diagnostic accuracy. The first study presents a magnetic bead-based approach for DNA microarray analysis for a rapid visual detection of single nucleotide polymorphisms. In the second work, magnetic beads were used as detection reagents for rapid differential detection of presence of pestiviral family members using a DNA oligonucleotide microarray with read-out by means of a tabletop scanner or a digital camera. In paper three, autoimmune responses from human sera were detected on a protein autoantigen microarray, again by means of magnetic bead analysis. Here, special emphasis was made in comprehensively comparing the performance of the magnetic bead detection to common fluorescence-based detection. In the fourth study, an immunochromatographic lateral flow protein microarray assay is presented for application in the classification of contagious pleuropneumonia from bovine serum samples. The analysis could be performed within 10 minutes using a table top scanner, and the performance of the assay was shown to be comparable to that of a cocktail ELISA. In the fifth paper, the lateral flow microarray framework is investigated in further detail by means of experiments and numerical simulation. It was found that downstream effects play an important role, and the results further suggest that the downstream binding profiles may find use in simple affinity evaluation.

Place, publisher, year, edition, pages
Stockholm: KTH, 2009. xii, 90 p.
Trita-BIO-Report, ISSN 1654-2312 ; 2009:18
magnetic microbeads, gold nanobeads, microarrays, lateral flow, on-site diagnosis, SNPs, pestivirus, autoimmunity, contagious bovine pleuropneumonia, comsol multiphysics, finite element method
National Category
Microbiology Medical Biotechnology (with a focus on Cell Biology (including Stem Cell Biology), Molecular Biology, Microbiology, Biochemistry or Biopharmacy)
urn:nbn:se:kth:diva-11221 (URN)978-91-7415-416-0 (ISBN)
Public defence
2009-10-23, FR4 Oskar Klein, AlbaNova Universitetscentrum, Roslagstullsbacken, Stockholm, 10:00 (English)
QC 20100713Available from: 2009-10-07 Created: 2009-10-07 Last updated: 2011-11-23Bibliographically approved

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