Location: Floral and Nursery Plants Research
Project Number: 8020-22000-042-11-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2019
End Date: Jul 31, 2021
The overall objective is the development of a CANARY potyvirus biosensor assay for the detection of potato virus Y (PVY) in potato; initially for assaying leaf samples and eventually for the detection of PVY in potato tuber tissue. Objective 1: Develop a ready-to-use biosensor assay kit at ARS, in collaboration with cooperator. Objective 2: Validate the potato leaf assay at four lab locations [ARS, Cooperator's lab, and two potato seed certification labs (in Colorado and Idaho)]. Objective 3: Test the assay on tuber tissue.
Phase 1: Assay development at ARS in collaboration with Cooperator. The MIT Potyvirus (POTY) biosensor cell line and the CANARY luminescence reaction reader (Zephyr) will be provided to USDA-ARS lab. ARS scientists will evaluate and verify the performance of the POTY biosensors using their collection of potyviruses and expressed potyviral coat proteins. Previous research indicates that key binding epitopes are not "exposed" enough or available for capture-binding by the biosensor cell-surface expressed antibody (PTY-1) using standard ‘virus’ extraction buffers. Further evaluations will now include testing/improving the extraction and antigen-antibody binding protocols, including use of unique buffers and various capture beads or membranes to "present" the POTY epitope to 'bridge' cell surface-expressed adjacent antibodies to initiate the biosensor cell signaling cascade. These assays will be developed using leaf tissue from various and diverse potyvirus-infected sources (including PVY isolates). When they become available, known healthy and virus-infected (primarily PVY) potato leaf samples will be sent to ARS and Cooperator from the two other project Cooperators (from Colorado and Idaho). These potato leaf samples will be determined and chosen from their annual testing of respective seed lot potatoes. PVY-infected (and selected healthy) plants will be flagged for eventual harvesting of tubers for testing in Phase 3. Standard ELISA-based reagents and protocols (including PTY-1) and virus-specific RT-PCR assays will be tested side-by-side to determine sensitivity, specificity and comparative usefulness. Extracts from healthy tubers from similar cultivars will be used as diluents to test potential inhibition in biosensor leaf assay reactions. The final protocol will be further developed in collaboration with the Cooperator into a user-friendly kit format for testing on-site in Colorado and Idaho. Phase 2: Leaf assay validation at four lab locations (ARS, Cooperator, and CO and ID cooperators). Cooperator and ARS will initially verify the protocol on 20 leaf samples of each of 4-5 cultivars sent from CO and ID, respectively, of field grown potatoes. Cooperator will then bring the potyvirus biosensors, set-up the biosensor equipment, and train the other cooperator users at both CO and ID. Both labs will test 40 leaf samples from 4-5 cultivars each. This assay validation and on-site training phase will assay a total of at least 480 samples. Phase 3: Tuber assay tests (ARS, CO and ID). Healthy and PVY-infected tubers (20-40 each from CO and ID cooperators) identified from the Phase 1 field surveys and assays will be the source of tuber tissue material to be sent to ARS for potyvirus biosensor assays on tuber extracts. Results will be compared to standard ELISA and RT-PCR assays with these tuber samples and leaf tissues from the grow-out samples.