1a.Objectives (from AD-416):
Develop a custom multiplex TaqMan® Array card as a standardized real-time polymerase chain reaction assay for simultaneous detection of RNA and DNA citrus pathogens of regulatory importance to the California citrus nurseries.
1b.Approach (from AD-416):
Use and modify commercial kits which capture nucleic acid on magnetic beads during robotic extraction to yield high quality total nucleic acid; utilize existing literature and validate or improve real-time PCR detection of specific RNA and DNA targets; develop a custom micro-fluidic card with multiple pathogen target signatures (primer sets) for amplicon reaction with TaqMan® probes; use a block specifically designed to conduct qPCR with 384-well microfluidic cards.
Results of 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. Quarantine and sanitary certification for California citrus nurseries require annual disease testing of >7,000 registered budwood source trees used for citrus propagation. Testing for pathogens causing exocortis or psorosis diseases of citrus is dependent on greenhouse biological indexing, a laborious process which limits testing to only ~800 trees per year. Currently, each tree is tested only once every 5-years. However, annual pathogen testing is required for tristeza and huanglongbing diseases. A real-time polymerase chain reaction (PCR) assay was modified to allow sensitive and simultaneous detection for multiple citrus pathogens in a 96-well plate. Thus, while testing for tristeza and huanglongbing, the same sample may be tested for additional pathogens in the same assay. Sampling, test duration and costs of the new assay are greatly reduced. Using the new assay, all citrus nursery source trees may be tested annually for all required pathogens in a standardized test. A version of this test has been certified by California Dept. of Food and Agriculture and has replaced biological indexing to test for citrus diseases caused by viroids. The general format used is versatile and can be bundled in detection kits adapted for multiple specified citrus pathogens. For example, a tristeza genotype PCR kit is now being tested by the Citrus Pest Detection Agency (aka, Central California Tristeza Eradication Agency, Tulare, California) to identify field trees infected by virulent strains of the virus causing tristeza stem pitting. Application of this technology facilitates identification of trees for removal much sooner than traditional assays.