Project Number: 2034-22000-015-004-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Aug 1, 2023
End Date: Sep 30, 2024
The objective is to select a suitable target (Citrus yellow vein clearing virus (CYVCV)-encoded polypeptide) to develop an antibody-based method capable of detecting CYVCV in the early stages of infection and to further develop this method into a lateral flow rapid test strips for CYVCV. The Cooperator will collect citrus field samples and use the test strip for detection of CYVCV and compare with Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR).
1. Citrus yellow vein clearing virus (CYVCV)-encoded proteins will be purified and used to produce polyclonal antisera. The complementary DNA sequence of CYVCV-encoded protein will be cloned into the bacterial protein expression vector pET28a. The recombinant plasmid will be transformed into Escherichia coli BL21 (DE3). The His-tagged proteins will be extracted and purified using HisTrap beads. 2. Efficiency of the polyclonal antisera for detection of CYVCV will be accomplished by testing CYVCV-infected trees with different polyclonal antisera at different time points (samples will be collected weekly post-inoculation). The CYVCV protein/polypeptide with the highest sensitivity will then be selected to generate the corresponding antibody. 3. Cooperator will collect sample tissue from field citrus trees and test for CYVCV by test strip and compare results with Enzyme-Linked Immunosorbent Assay (ELISA) and Reverse Transcription Quantitative Polymerase Chain Reaction (RT-qPCR).