Location: Vegetable Research
Project Number: 6080-22000-032-012-R
Project Type: Reimbursable Cooperative Agreement
Start Date: Oct 1, 2023
End Date: Aug 31, 2025
Objective:
1. To develop genera-specific RPA primer probes and establishing the assay protocol against common cucurbit virus genera including Begomovirus, Potyvirus, Cucumovirus, Tobamovirus, Crinivirus, Coguvirus, and Carmovirus and test for sensitivity and specificity.
2. To develop species-specific RPA primer probes to major cucurbit viruses including CGMMV, CMV, WCLaV-1, WCLaV-2, WSMoV and MNSV and test for sensitivity and specificity.
3. To standardize genera and species-specific RPA assays detection from seeds, leaves, vines, fruits, flowers, roots, and pollen.
4. To transfer RPA technology to a field usable product: Designing and creating an easy-to-handle cucurbit RPA assays toolbox.
5. Validation of one-step RPA assays toolbox in regional plant virology laboratories in the southeastern U.S and training for lab personnel, and stakeholders and creation of new web-based education platform for RPA use.
Approach:
Relevant to objectives 1 and 2: The genus-specific degenerate primers for RPA assay will be designed to collect complete genomic sequences of each genus available in the NCBI GenBank database and aligned through multiple sequences alignment in Bioedit software. A pair of polyvalent primers will be identified to target these viruses' conserved region of the replicase gene or coat protein gene (CP) of following genera: Begomovirus, Carmovirus, Coguvirus, Crinivirus, Cucumovirus, Potyvirus, Tobamovirus, and Tospovirus. The species-specific primers also designed above said concept and the feasibility of the genus-specific degenerate and species-specific primer sequences for 13 selected viruses will be evaluated by the oligonucleotides properties calculator at integrated DNA Technologies (IDT). The genus-specific degenerate probes will be designed according to the same protocol following the condition of the degenerate primer. Fluorescence detection in the FAM channel (excitation 470 nm and detection 520 nm) will be performed using an Amplifire (Agdia Inc) and Axxin T8-ISO at 39C for 30 minutes (Twist DX, Cambridge, UK). All results generated using either type of RPA will be verified using polymerase chain reaction with degenerate primers.
