2012 Annual Report
1a.Objectives (from AD-416):
To develop and validate a universal plant virus microarray for detection and differentiation of plant viruses. To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts.
1b.Approach (from AD-416):
ARS and the Cooperator will assemble the lists of viral taxa to be represented on the viral detection microarray, and virus-infected samples from which to amplify nucleic acids to validate the microarray. This information and material will be utilized by both ARS and the Cooperator to jointly develop and validate the microarray for detection of target viruses, and to make validation results available to collaborators via a web server. The Cooperator will perform analysis of viral sequences to identify suitable sequences for the development of oligonucleotides, supervise production of the microarrays based on the selected oligonucleotides, perform assays to validate the array with as many viruses as possible from the collections of all cooperators, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples.
Additional print runs of the Universal Plant Virus Microarray (UPVM) were made at Washington University. Then, following the acquisition of a microarray printing system at the Danforth Plant Science Center, assistance was provided for assembly and extensive calibration of the arrayer; subsequently the printing at the Danforth Center of test runs of the UPVM used the new arrayer. In order to reduce print run times, further arrays were printed in 32 blocks (8 rows x 4 columns) rather than the previous 16 blocks, cutting the time per print run of 261 slides from approximately 22 hours to about 11 hours.
To date several hundred production UPVM slides have been printed and distributed to the various UPVM collaborating laboratories, where hybridization for validation and other testing of the UPVM is being carried out (USDA-ARS in Beltsville, MD; the Danforth Center in St. Louis. MO; Oklahoma State University, Stillwater, OK; and, Cornell University, Ithaca, NY). Analysis of the hybridizations performed by the collaborators is occurring through use of software developed by a UPVM collaborator at the University of Utah. This information will be of most immediate application to the UPVM collaborators, but will also be of value to regulatory agencies, plant diagnostic clinics, germplasm repositories, and producers operating plant certification schemes.