|Yokomi, Raymond - Ray|
Submitted to: Meeting Abstract
Publication Type: Abstract Only
Publication Acceptance Date: 3/30/2006
Publication Date: 7/16/2006
Citation: Lin, H., Doddapaneni, H., Civerolo, E.L., Yao, J., Yokomi, R.K., Bai, X., Cao, H., Zhao, X., Wen, R. 2006. Multiplex taqman-based pcr for sensitive and accurate quantification of citrus hlb and cvc pathogens; candidatus liberibacter and xylella fastidiosa [Abstract]. Proceedings of the Huanglongbing-greening International Workshop. p. 83.
Technical Abstract: Citrus Huanglongbing (HLB, also known as citrus greening disease) and citrus variegated chlorosis (CVC) are caused by Candidatus Liberibacter (CL) and Xylella fastidiosa (Xf) respectively. Both pathogens were listed as plant bio-threat agents in 2002 US Agricultural Bioterrorism Protection Act. Citrus HLB is one of the most devastating epidemic diseases in southern parts of China as well as in tropical and subtropical regions worldwide. The recent confirmation of HLB in Florida, USA (September 2005) has increased threats to the US citrus industry. 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 pathogen. We outline here a strategy for the generation of a multiplex Taq-Man PCR assay that was designed to monitor both CL-HLB and Xf-CVC levels in a single assay. We developed a bioinformatics pipeline to select targeted loci for high quality and specificity primer and probe designs. The algorithm evaluated sequences from selected primers and probes to generate primer/probe sets with the least interactions for multiplex reactions. Specificities were validated by in silico BLAST and PCR tests with citrus-associated pathogens and citrus DNAs. This detection system in combination with our sample preparation protocol was optimized for effective, sensitive and reliable assaying clinical samples.