A DNA oligonucleotide microarray for detecting human astrovirus serotypes

Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of l...

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Bibliographic Details
Published in:Journal of virological methods 2008-01, Vol.147 (1), p.86-92
Main Authors: Brown, David W., Gunning, Kerry B., Henry, Dorothy M., Awdeh, Zuheir L., Brinker, James P., Tzipori, Saul, Herrmann, John E.
Format: Article
Language:English
Subjects:
Astrovirus
Biological and medical sciences
Caco-2 Cells
DNA oligonucleotide microarray
Feces - virology
Fundamental and applied biological sciences. Psychology
Human astrovirus
Human astroviruses
Humans
Mamastrovirus - classification
Mamastrovirus - isolation & purification
Microbiology
Nucleic Acid Hybridization
Oligonucleotide Array Sequence Analysis - methods
Oligonucleotide Probes
Reverse Transcriptase Polymerase Chain Reaction
RT-PCR
Serotyping
Techniques used in virology
Virology
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Summary:Human astroviruses have been shown in numerous studies to be an important cause of gastroenteritis in young children worldwide. The present communication addresses their characterization by use of oligonucleotide microarray hybridization. The system developed consists of an RT-PCR using primers of low degeneracy capable of detecting all eight serotypes of human astroviruses. RT-PCR products are then hybridized against a microarray consisting of short oligonucleotide probes 17–18 nucleotides in length. Cy3-labeled ssDNA targets are generated using a Cy3-labeled primer in the RT-PCR. The non-labeled strand is enzymatically digested, and the labeled target is rescued by column purification. This method of generating labeled target uses equimolar concentrations of the amplifying primers and does not compromise assay sensitivity for initial detection of the virus. Hybridization can be performed without the need for additional amplification. Although the amplicon spans a relatively conserved region of the astrovirus genome, the use of short probes enables type distinction despite such limited diversity. Probes differing by as little as a single nucleotide can be used to distinguish isolates. The microarray developed was capable of distinguishing representatives of the eight known serotypes of human astroviruses.
ISSN:0166-0934
1879-0984
DOI:10.1016/j.jviromet.2007.07.028