The primary purpose of this agreement is the consolidation of 10 singleplex qpcr assays currently in use by the california citrus clonal protection program (ccpp), into three multiplex qpcr assays and…

The U.S. Department of Agriculture's Animal and Plant Health Inspection Service (APHIS) awarded a $143,072 Cooperative Agreement under the Plant and Animal Disease, Pest Control, and Animal Care Federal Grant Program (CFDA 10.025) to the Regents of the University of California at Riverside. The primary purpose of this agreement is to consolidate 10 singleplex real-time PCR (qPCR) assays currently used by the California Citrus Clonal Protection Program (CCPP) into three multiplex qPCR assays, and to redesign one singleplex qPCR assay for an emerging citrus virus, Citrus Yellow Vein Clearing Virus (CYVCV). The 10 targeted citrus pathogens include two prokaryotes, two DNA viruses, one RNA virus, and five viroids. The newly developed assays will be experimentally validated and disseminated to interested diagnostic laboratories in California and other major citrus-producing regions of the U.S. This project aims to improve diagnostic capabilities for citrus pathogens of regulatory importance, which will benefit federal and state agricultural agencies as well as citrus growers.

The primary purpose of this agreement is the consolidation of 10 singleplex qpcr assays currently in use by the california citrus clonal protection program (ccpp), into three multiplex qpcr assays and the redesign (based on updated pathogen genome sequences) of one singleplex qpcr assay for an emerging citrus virus threatening the ccpp germplasm sources namely, citrus yellow vein clearing virus (cyvcv). The developed assays will target the following 10 citrus pathogens: cyvcv, chlorotic dwarf-associated virus (ccdav), citrus yellow mosaic virus (cymv), citrus bent leaf viroid (cblvd), citrus dwarfing viroid (cdvd), citrus viroids v (CVD-v), -VI, and -VII, 'candidatus liberibacter SP.', and spiroplasma citri. Activities to be performed: the main goal is to design and validate three multiplex and one singleplex qpcr detection assay targeting 10 high-risk citrus pathogens of regulatory importance. The targeted pathogens include two prokaryotes, two DNA viruses, one RNA virus, and five viroids. To achieve the project goal, new assays will be developed from current assays. The newly developed assays will then be experimentally validated using established performance criteria so they can be used reliably for screening citrus for pathogens of concern. Successful completion of the proposed project will lead to successful consolidation of multiple diagnostic tests, currently used as singleplex detection format into the multiplex assay format, hence reducing time and resources. Deliverables from this project include: four newly designed qpcrbased assays targeting 10 selected citrus pathogens of regulatory importance in california; dissemination of the new qpcr assays to interested diagnostic laboratories in california and other citrus-producing regions in the U.S., such as arizona, texas, hawaii, and puerto rico; cross-institutional qpcr assay validation adhering to miqe guidelines (transferability and repeatability); a written assay validation SOP which is to be the standard for future assay validation proceedings; at least one peer-reviewed and one semi-technical extension publication describing the project and our findings. By improving diagnostic test for citrus this project will benefit: USDA-aphis plant germplasm quarantine program; california department of food and agriculture; citrus growers in california and other citrus-producing RE gions in the U.S., such as arizona, texas, hawaii, and puerto rico.