Project Number: 5302-22000-011-04
Project Type: Reimbursable

Start Date: Oct 01, 2010
End Date: Jun 30, 2013

Objective:
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).

Approach:
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. Documents Reimbursable from the CA Dept of Food and Agriculture, 2010 Specialty Crop Block Grant Program. Log 41977.