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Simultaneous genotyping of all hemagglutinin and neuraminidase subtypes of avian influenza viruses by use of padlock probes
Uppsala University, Disciplinary Domain of Medicine and Pharmacy, Faculty of Medicine, Department of Genetics and Pathology, Molecular tools.
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2008 (English)In: Journal of Clinical Microbiology, ISSN 0095-1137, E-ISSN 1098-660X, Vol. 46, no 5, 1747-1751 p.Article in journal (Refereed) Published
Abstract [en]

A subtyping assay for both the hemagglutinin (HA) and neuraminidase (NA) surface antigens of the avian influenza virus (AIV) has been developed. The method uses padlock probe chemistry combined with a microarray output for detection. The outstanding feature of this assay is its capability to designate both the HA and the NA of an AIV sample from a single reaction mixture. A panel of 77 influenza virus strains was tested representing the entire assortment of the two antigens. One hundred percent (77/77) of the samples tested were identified as AIV, and 97% (75/77) were subtyped correctly in accordance with previous examinations performed by classical diagnostic methods. Testing of heterologous pathogens verified the specificity of the assay. This assay is a convenient and practical tool for the study of AIVs, providing important HA and NA data more rapidly than conventional methods.

Place, publisher, year, edition, pages
2008. Vol. 46, no 5, 1747-1751 p.
National Category
Medical and Health Sciences
Identifiers
URN: urn:nbn:se:uu:diva-16412DOI: 10.1128/JCM.02292-07ISI: 000255678200027PubMedID: 18353937OAI: oai:DiVA.org:uu-16412DiVA: diva2:44183
Available from: 2008-05-22 Created: 2008-05-22 Last updated: 2017-12-08Bibliographically approved
In thesis
1. Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation
Open this publication in new window or tab >>Ligation-mediated Molecular Analysis of Influenza Subtypes, Splicing and Protein Glycosylation
2010 (English)Doctoral thesis, comprehensive summary (Other academic)
Abstract [en]

Binder-based assays are employed throughout the life sciences. Powerful signal amplification techniques have enabled detection of very rare molecule species diluted in simple buffers. Unspecific binding of primary binders leads to increased background in more complex samples. By requiring two recognition events, ligation-based molecular analyses provide highly specific detection of biomolecules in complex samples.

We developed a highly multiplexed padlock-ligation assay targeting signature sequences in the hemagglutinin and neuraminidase genes. From a panel of 77 avian influenza isolates of all major serotypes, 97% were genotyped correctly in accordance with previous classifications by classical diagnostic methods (Paper I).

Alternative splicing is an important mechanism expanding the proteome. Current analysis techniques fail to provide sequences of complete transcripts beyond the read length of sequencing instruments. We devised and implemented a strategy to compress the sequence information contained in the splicing pattern of a transcript into the presence or absence of sequence-blocks. We demonstrate that this assay yields information about the splicing patterns in thousands of transcripts from cellular cDNA (Paper II).

Expression changes of mucin proteins and glycosylation structures are frequently observed from the early stages of cancer development. Expression of mucin 2 and sialyl-Tn are common features of intestinal metaplasia and gastric cancer, and are known to co-locate. Here we have developed an in situ proximity ligation assay (PLA) directed against mucin 2 and sialyl-Tn. Our study on intestinal metaplasia and gastric cancer tissue sections identified mucin 2 as a major carrier of sialyl-Tn in these conditions, and demonstrated how conveniently glycosylation of proteins can be studied by in situ PLA (Paper III).

This thesis shows how the dual recognition requirement of ligation-based assays can be employed to detect target molecules with high specificity, to analyze several sequence features of nucleic acids or to study the proximity of two antigens in situ.

Place, publisher, year, edition, pages
Uppsala: Acta Universitatis Upsaliensis, 2010. 39 p.
Series
Digital Comprehensive Summaries of Uppsala Dissertations from the Faculty of Medicine, ISSN 1651-6206 ; 532
Keyword
ligase, proximity ligation, gastric cancer, glycosylation, alternative splicing, avian influenza, padlock probe
National Category
Medical Genetics
Research subject
Molecular Medicine
Identifiers
urn:nbn:se:uu:diva-120016 (URN)978-91-554-7743-1 (ISBN)
Public defence
2010-04-23, Rudbecksalen, Rudbeck Laboratory, Dag Hammarskjölds Väg 20, Uppsala, 09:15 (English)
Opponent
Supervisors
Available from: 2010-04-01 Created: 2010-03-04 Last updated: 2010-04-01Bibliographically approved

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Conze, TimNilsson, MatsLandegren, Ulf

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