2013 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.
This is the final report for this project. Significant results were realized over the life of this project, including the demonstration of proof of principle using a 750-element ‘MiniPlantViroChip’ with probes for approximately 50 well-characterized plant viruses as well as control host genes. The ‘MiniPlantViroChip’ was based on oligonucleotides previously designed for a panviral microarray. In initial testing of the ‘MiniPlantViroChip’, all but one virus tested was successfully detected with a majority of the virus-specific oligonucleotides, without amplification of the samples. The tested viruses represented viruses with both RNA and DNA genomes, including members of the families Geminiviridae (single-stranded DNA genome) and Caulimoviridae (double-stranded DNA genome), as well as species in the Bromoviridae, Closteroviridae, Flexiviridae, Potyviridae, and Virgaviridae (all with single-stranded RNA genomes), including multiple strains of some viruses. These results demonstrated the specificity and sensitivity of the array over a wide dynamic range of probe intensities, and formed a strong background for development of the full scale Universal array. A total of 9,600 oligonucleotides were subsequently designed, with the theoretical capability to detect and identify all plant viruses and viroids for which complete genome information was available in public databases as of December 2009, and to identify previously uncharacterized viruses to the family and genus level in cases where availability of multiple sequences allowed design of family- and genus-level probes. Several hundred 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 Donald Danforth Center in St. Louis, MO; Oklahoma State University, Stillwater, OK; and Cornell University, Ithaca, NY). Assistance was provided for preparation and printing of additional UPVM slides at the Danforth Plant Science Center, with arrays printed in 32 blocks (8 rows x 4 columns) to reduce the print run time of 261 slides to about 11 hours. 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.