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. Documents Reimbursable from the CA Dept of Food and Agriculture, 2010 Specialty Crop Block Grant Program. Log 41977.
Results from this study are in support of Objective 1.A of the parent project. Target genome sequences were identified for important citrus pathogens. A nucleic acid hybridization assay using labeled microspheres and fluorescent signal amplification was developed which allowed simultaneous detection of 9 citrus pathogens or strains and an internal citrus gene target as a control. Sensitivity and linearity of the assay was equivalent to that of enzyme-linked immunosorbent assay for each of the different pathogens in a dilution range from 1 to 1000. Another technique, which combines hybridization with polymerase chain reaction amplification from the target sequence (MOL-PCR) was used successfully with synthetic citrus pathogen targets but problems were encountered in testing extracts from infected citrus plants. Research on both methods are continuing.