Develop high throughput extraction procedures for RNA and DNA citrus pathogen targets for multiplex MOL-PCR assays
Location: Crop Diseases, Pests and Genetics
Project Number: 5302-22000-011-05
General Cooperative Agreement
Start Date: Jan 01, 2011
End Date: Jun 30, 2013
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.
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.