2012 Annual Report 1a.Objectives (from AD-416): Develop cost-effective and highly sensitive methods to detect huangblongbing (HLB) and multiplex it with detection of other regulated pathogens, thus, improving the cost/benefit ratio per marker per pathogen being diagnosed. 1b.Approach (from AD-416): 1. Establish collections of citrus pathogens including CTV, CVV (Xylella fastidiosa), CPsV, Pospiviroids (CEVd, CVd-IV), Hostuviroids (CVd-IIa, CVd-IIb, CVd-IIc), stubborn (Spiroplasma citri), huanglongbing (HLB, “Candidatus” Liberibacter asiaticus, “C.” L. africanus), Leprosis, and Citrus leaf blotch virus (Dweet mottle) and Tatterleaf; 2. Establish reliable real-time PCR detection of these pathogens; 3. Establish rapid extraction of pathogen nucleic acid from target tissue; 4. Use results of computational design for oligonucleotides for specific identification of the panel of citrus pathogens from LANL collaborators, assemble fluorescent dye to oligo markers and employ Luminex instrument for detection by MOL-PCR; and 5. Test automated high-throughput nucleic acid extraction machines for target oligo acquisition from citrus tissue. 3.Progress Report: Results of this study are in support of Objective 1.A of the parent project. The goal of this project is to develop efficient and cost-effective methods to sample citrus to detect infection by citrus pathogens, or strains, in one simultaneous assay. A procedure for high throughput, robotic sample processing was developed that resulted in high quality nucleic acid (RNA and DNA) samples from citrus tissue infected with various citrus pathogens. These nucleic acid samples contain pathogen DNA or RNA which serve as targets for use in innovative DNA-based sequence-specific assays. A hybridization assay was developed based on binding of specific pathogen target sequences to labeled microspheres with a built-in DNA signal amplifier. The assay is read in a flow cytometer instrument equipped with lasers and a detector to read fluorescence of the signal amplifier. This assay was validated for the simultaneous detection of 9 different citrus pathogens and a citrus gene target as an internal control.