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 (Develop field deployable systems that provide rapid, sensitive detection of Citrus tristeza virus and Spiroplasma citri in citrus) of the in-house project. After thorough evaluation of a technique of Multiplexed Oligonucleotide Ligation–PCR (MOL-PCR) using in planta citrus tissue infected with various citrus pathogens, with research partner, Los Alamos National Laboratory, developer of the MOL-PCR technology, the procedure was found to still require further improvements before it can be used reliably to detect citrus pathogens from field samples. Therefore, other lines of research were explored to accomplish research objectives. Improved efficiency with reduced costs for pathogen surveillance is essential for detection or interdiction of exotic pathogens. Pathogen nucleic acids were purified from citrus tissue extracts in an automated, high throughput instrument using a magnetic bead-based commercial kit modified for detection of RNA and DNA targets. These pathogen targets were used to detect up to 10 different pathogens or strains simultaneously in a single assay using either a polymerase chain reaction (PCR) or hybridization assay. Both platforms use sequence-specific nucleic acid targets and are easily adapted for different pathogens. Both protocols have great flexibility to detect complexes of pathogens. This feature allows great flexibility to detect complexes of pathogens using standard laboratory instruments measuring fluorescence or a flow cytometer. A high throughput real-time PCR assay was certified by the California Department of Food and Agriculture this year for certification of viroid-free citrus nursery stock. Previously, a bioindex test in Citrus medica (citron) seedlings was used. This bioassay requires a nine-month incubation period in a greenhouse, limiting testing of a budwood scion source to once every five years. Using this new multiplexed lab-based diagnostic assay, viroid testing may be performed annually.