Submitted to: Phytopathology
Publication Type: Abstract Only
Publication Acceptance Date: 3/30/2006
Publication Date: 7/29/2006
Citation: Lin, H., Doddapaneni, H., Yao, J., Civerolo, E.L. 2006. Multiplex TAQ-MAN PCR for Sensitive and Accurate Quantification Bio-threat Crop Pathogens; Xylella fastidiosa and candidatus, liberibacter. Phytopathology. 96:S127. Interpretive Summary:
Technical Abstract: Citrus variegated chlorosis (CVC) and citrus Huanglongbing (HLB, also known as citrus greening disease) are caused by Xylella fastidiosa (Xf) and Candidatus, Liberibacter (CL) respectively. CVC and HLB are severely affecting citrus production in tropical and sub-tropical regions of the world. To effectively prevent serious economic losses, ensure a safe and sustainable agriculture, highly sensitive and specific detection tools are needed for early and accurate detection of the pathogens. We outline here a strategy for the generation of multiplex Taq-Man PCR assays that were used to monitor both Xf-CVC and CL-HLB levels in a single assay. We developed a bioinformatics pipeline to select targeted loci for high quality and specificity primer and probe designs. Specificities were validated by in silico BLAST and PCR tests with citrus-associated pathogens and host DNAs. We refined our nucleic acid extraction procedures for reliable clinical sample assays. The multiplex assay system directly measures the absolute number of pathogen copies in clinic samples.