1a.Objectives (from AD-416):
To demonstrate the ability of an oligonucleotide microarray to detect and differentiate plant viruses from random amplification of plant total nucleic acid extracts, and to provide education, through training, of undergraduate interns, and the incorporation of macroarray and microarray techniques into curricula.
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, and participate in analysis of microarray hybridization results to determine with a high degree of confidence which viruses were present in validation samples. The Cooperator will also integrate macroarray and microarray techniques into curricula, and educate students on the theory and application of macroarrays and microarrays.
In the design and fabrication of a macroarray for the detection of grapevine viruses, all virus- and genera-specific oligonucleotide probes designed for the UPVM array and specific for viruses infecting grapevine were included in the macro array format. Host plant-specific probes were also included. A method to enhance the sensitivity of array-based virus detection is being developed, based on hybridization of ribosomal RNAs to capture probes and removal using magnetic beads. The depletion of ribosomal RNAs has been demonstrated by agarose gel analysis of stainable RNA, and by enhanced detection of grapevine viruses in a macroarray format. Probes for virus detection were shown to be effective in detection of two viruses previously unreported in the northeastern United States. This work has resulted in two publications that are in press in peer-reviewed journals. 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.