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 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.
Brown
David W
DW
Tufts University, Cummings School of Veterinary Medicine, North Grafton, MA 01536, USA. David.Brown@Tufts.edu
Gunning
Kerry B
KB
Henry
Dorothy M
DM
Awdeh
Zuheir L
ZL
Brinker
James P
JP
Tzipori
Saul
S
Herrmann
John E
JE
D018938:Caco-2 Cells; D005243:Feces / Q000821:virology; D006801:Humans; D001257:Mamastrovirus / Q000145:classification / Q000302:isolation & purification*; D009693:Nucleic Acid Hybridization; D020411:Oligonucleotide Array Sequence Analysis / Q000379:methods*; D015345:Oligonucleotide Probes; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012703:Serotyping
D015345:Oligonucleotide Probes