Articles
PROGRESS IN THE DEVELOPMENT OF A UNIVERSAL PLANT VIRUS MICROARRAY FOR THE DETECTION AND IDENTIFICATION OF VIRUSES
Article number
1072_18
Pages
149 – 156
Language
English
Abstract
Microarrays based on oligonucleotides representing sequences conserved at the level of viral species, genera, and families are able to detect and identify both characterized and previously uncharacterized viruses infecting mammals and birds.
Software initially developed for these animal virus microarrays has been further refined for both design and analysis of a Universal Plant Virus Microarray (UPVM). The UPVM is based on 9,600 60-mer oligonucleotides, including at least four genus-level and four family-level probes per taxonomic group, and 44 control probes for highly conserved plant genes.
These probes together represent all characterized plant viruses for which significant genomic sequence was publically available in GenBank as of January 2010, and additional sequences made available to us prior to public GenBank release.
Associated methods have been developed for high quality total nucleic acid extraction, applicable to a broad range of plant tissues containing metabolites such as phenolics, polysaccharides, latex, and resins that can interfere with nucleic acid extraction or subsequent amplification.
Validation of the UPVM with a broad range of DNA and RNA plant viruses is in progress.
Many high-titer viruses can be detected by direct labeling of total RNA extracts.
Amplification and subtractive hybridization protocols to increase the sensitivity of detection of low titer viruses are being examined.
Software initially developed for these animal virus microarrays has been further refined for both design and analysis of a Universal Plant Virus Microarray (UPVM). The UPVM is based on 9,600 60-mer oligonucleotides, including at least four genus-level and four family-level probes per taxonomic group, and 44 control probes for highly conserved plant genes.
These probes together represent all characterized plant viruses for which significant genomic sequence was publically available in GenBank as of January 2010, and additional sequences made available to us prior to public GenBank release.
Associated methods have been developed for high quality total nucleic acid extraction, applicable to a broad range of plant tissues containing metabolites such as phenolics, polysaccharides, latex, and resins that can interfere with nucleic acid extraction or subsequent amplification.
Validation of the UPVM with a broad range of DNA and RNA plant viruses is in progress.
Many high-titer viruses can be detected by direct labeling of total RNA extracts.
Amplification and subtractive hybridization protocols to increase the sensitivity of detection of low titer viruses are being examined.
Authors
J. Hammond, D.C. Henderson, B. Bagewadi, R.L. Jordan, K.L. Perry, U. Melcher, D. Wang, K.F. Fischer, C.M. Fauquet
Keywords
hybridization, T-predict, nucleic acid extraction, unbiased amplification, uncharacterized virus
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