1a. Objectives (from AD-416)
Develop multiplex assays based on two PCR technologies: i) real-time reverse transcription (RT)-PCR using fluorescent probes; ii) multiplex oligonucleotide ligation-PCR (MOL-PCR) followed by a bead-based revelation system (Luminex).
1b. Approach (from AD-416)
Gene sequence data of key regulatory citrus pathogens in pubic database has resulted in development of molecular markers designed from generic to specific pathogen strain detection in polymerase chain reaction (PCR) assays. Real time PCR allows target quantification and will be used to determine seasonal optimum for pathogen detection. Total nucleic acid capture and purification will be used as templates for both DNA and RNA pathogens. Detection will be multiplexed by detection of single nucleotide polymorphisms (SNPs) and their presence or absence. Multiple pathogens or pathogen strains can be easily combined in a single assay by Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). Ligation to universal primers occur at high temperature, PCR conducted with universal primers and specific fluorescent microsphere (bead) by Luminex Instrument capture and analysis. Computational design identifies target genes, conducts phylogeny and determines canonical SNPs. Design of SNP-specific MOLioges will be performed by MOLiogDesigner Tool which checks MOLigo pair robustness and provides multiplex analysis. Multiplex detection will be validated by singleplex target detection by PCR or ELISA in inclusivity/exclusivity panel testing.
3. Progress Report
This report documents research conducted under a Reimbursable Agreement in support of Objective 3 of the parent project, 5302-22000-009-00D. The goal is to develop rapid diagnosis of pathogens spread to citrus by vectors or propagation. Target genes will be identified using computational design to conduct phylogeny and determine canonical SNPs. SNP-specific MOLioges will be developed by MOLiogDesigner Tool which checks MOLigo pair robustness. Pathogen detection is proposed to be performed in a single assay by Multiplex Oligonucleotide Ligation-PCR (MOL-PCR). Multiplex detection will be validated by singleplex detection by polymerase chain reaction (PCR) or enzyme-linked immuno-sorbent assay (ELISA). This research will be conducted by an ARS-Parlier-led team with collaborators at UC Riverside and the Los Alamos National Laboratory. Delay in establishing collaborative agreements have contributed to the late start of this research.