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Title: Progress in development of a Universal Plant Virus Micrarray for the detection and identification of plant viruses

item Hammond, John
item Henderson, David
item BAGEWADI, BASAVARAJ - Danforth Plant Science Center
item Jordan, Ramon
item PERR, KEITH - Cornell University
item MELCHER, ULRICH - Oklahoma State University
item WANG, DAVID - Washington University
item FISCHER, KAEL - University Of Utah
item FAUQUET, CLAUDE - Danforth Plant Science Center

Submitted to: Acta Horticulturae
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 1/21/2014
Publication Date: 2/21/2015
Citation: Hammond, J., Henderson, D.C., Bagewadi, B., Jordan, R.L., Perr, K., Melcher, U., Wang, D., Fischer, K., Fauquet, C. 2015. Progress in development of a Universal Plant Virus Micrarray for the detection and identification of plant viruses. Acta Horticulturae. 1072:149-156.

Interpretive Summary: Most traditional virus detection methods require a specific test to be performed for each particular virus or group of viruses to be identified, and thus require many individual assays in order to test for a large number of viruses. These tests may require distinct extraction and assay methods, as well as virus-specific reagents for each type of virus. Microarray technology offers a means of applying a single method to test for many different viruses simultaneously, with the potential ability to detect and identify all components of a mixed infection. Progress in the development and validation of a Universal Plant Virus Microarray, intended to be able to detect and identify almost any currently recognized plant virus or viroid, is described, together with associated methods for sample preparation. The Universal Plant Virus Microarray is also anticipated to be able to identify previously uncharacterized viruses to the level of viral genus or family, thus providing information about possible modes of transmission. Availability of a universal method for plant virus detection and identification would greatly simplify the task of plant diagnostic clinics, and potentially allow earlier application of appropriate control measures to minimize introduction and spread of newly emergent viruses into agricultural and horticultural production.

Technical 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 9600 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.