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 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.
The first set of Universal Plant Virus Microarray (UPVM) slides were printed in a core facility at Washington University at St. Louis, MO. An additional arrayer to print UPVM slides was obtained and re-assembled at the Danforth Plant Science Center. After extensive calibration and several test prints, the arrayer was used to print three additional sets of UPVM slides using the same set of 60-mer probes aliquoted in 25 384-well plates. In order to reduce the printing time from c. 22 hours to c. 11 hours, additional sets were printed in 32 blocks (8 rows x 4 columns) rather than 16 blocks. A total of 783 slides were printed, tested for quality, and distributed to the collaborating labs. The validation of UPVM was continued using the new print sets and the appropriate data acquisition file for the array readers. High titer viruses could easily be detected and identified without the amplification of starting nucleic acid. For low-titer virus samples several strategies including increasing the quantity of total RNA for labeling, depletion of host rRNAs and random-amplification are being tested. Increasing the total RNA for labeling and pooling two labeled reactions before hybridization resulted in the detection some viruses (e.g., Uganda cassava brown streak virus, Ourmia melon virus) that were not detected by the original protocol. Removal of rRNA, which constitutes about 90% of total RNA, was examined as a means to enhance sensitivity; initial results suggest that a microsphere based method (Ribozero kit) worked more efficiently than a streptavidin-coated magnetic beads method (Ribominus kit); however these are very expensive. An mRNA 5’cap binding protein (GST-eIF4E) from N. benthamiana has been cloned and overexpressed in E. coli and will be used with biotinylated oligo-dT to select mRNAs with 5’ cap and /or 3’ polyA tail.
In a majority of cases identification of the virus(es) by the hybridization intensity of the probes was in agreement with T-Predict (an improved version of E-predict, developed at the University of Utah lab). In order to further enhance the predictive power of the T-predict, UPVM slides were hybridized with both diseased and healthy samples of Grapevine, Peanut, Sweet potato, Brachypodium, Hibiscus and Citrus. Comparison of hybridization profiles of healthy and infected plant samples identified non-specific reactions of a few of the 60-mer probes and aided interpretation of the UPVM results.
Microarray data was also analyzed by hierarchical clustering and T-Predict to identify one or more viruses in artificially mixed virus samples. Some of these results 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 talk “All plant virus chip: shifting from proof to use” was presented in a special session on Advances in Detection Technologies at the American Phytopathological Society meeting in August 2012. 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.