2011 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 will acquire 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, provide the facilities for production of the microarrays based on the selected oligonucleotides,and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples.
Prior development and validation of a demo/prototype DNA microarray platform (the MiniPlantViroChip) has been followed by the development of a complete version of the Universal Plant Virus Microarray (UPVM). The full UPVM has been tested with 22 plant virus samples from infected plant extracts. We have successfully utilized a predictive computational algorithm (developed by a University of Utah collaborating scientist) to analyze and interpret the UPVM hybridization data. Fifteen of 22 viruses were successfully detected and correctly identified without amplification of the samples. The viruses successfully detected include several members of the Geminiviridae (ssDNA genomes), and members of the Potyviridae, Alphaflexiviridae, Betaflexiviridae, Bromoviridae, and Virgaviridae (all positive-sense ssRNA genomes), as well as Tomato spotted wilt virus (Tospovirus, Bunyaviridae, ambisense RNA genome). Other samples of geminiviruses, potyviruses, and one tobamovirus were not detected, possibly due to low titer. Additional samples from various healthy host plants confirmed the utility of the control host plant probes and absence of significant non-specific host reactions to virus-specific probes. Validation with other viruses, satellites and viroids and the assembly of an in-house microarray printing robot are in progress. The UPVM should provide an economical tool to screen any biological material for any known viruses, or unknown virus related to the known viruses, without requiring prior knowledge of the virus(es) present, and in a relatively short time (i.e., 2-3 days). However, the challenges include extraction of good quality nucleic acid from the host plants, and detection of the viruses with low titer infection. We have shared with our collaborators the aliquots of the 9600 oligonucleotides, fabricated UPVM slides and protocols and other reagents. We are contributing and participating in development of a training workshop for stakeholders, to be held in November 2011, involving all of the UPVM collaborators and stakeholders.
Communications to monitor progress were carried out by e-mail and either individual or conference calls between the various partners, by a meeting during the Annual meeting of the American Phytopathological Society, and by written and oral reports to the NRI Plant Biosecurity Program.