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 Universal Plant Virus Microarray (UPVM), and specific for viruses infecting grapevine, were included in the macro array format. Host plant specific probes were also included. Some of the plant probes proved useful as internal controls showing that they hybridized to grapevine sequences. Very few of the UPVM virus probes showed hybridization to extracts prepared from leaf roll infected grapevines. RNA extracts prepared from virus infected grapevines and tested on the macro array were sent to the lab at the Danforth Plant Science Center for testing on the UPVM. Results obtained with plant virus macroarrays were presented at the UPVM Workshop and BARD workshop ‘Microarrays and Next-Generation Sequencing for Detection and Identification of Plant Viruses’ held at Beltsville in November 2011. A publication “Macroarray detection of grapevine leafroll associated viruses” was published in the Journal of Virological Methods (183: 161-169). 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.