|Yokomi, Raymond - Ray|
Submitted to: American Phytopathological Society Abstracts
Publication Type: Abstract Only
Publication Acceptance Date: 6/12/2007
Publication Date: 7/1/2007
Citation: Saponari, M., Yokomi, R.K. 2007. Real-time RT-PCR Assay for Detection and Differentiation of Citrus Tristeza Virus Isolates. Phytopathology. 97:S104.
Technical Abstract: Multiplex one step real time RT-PCR assays using TaqMan probes were developed for detection and strain differentiation of Citrus tristeza virus (CTV). For broad spectrum CTV detection, a TaqMan primer and Cy5-labeled probe were designed using CP gene sequences. An internal control was developed using sequences from the plant mitochondrial nad5 gene to assess the quality of the total RNA extracted. Nucleotide sequence alignments between the major and minor CP gene regions of various CTV isolates revealed that stem pitting and seedling yellows isolates share a conserved sequence which differs from other isolates. This sequence was used to design a specific TaqMan primer and FAM-labeled probe to distinguish VT genotype and seedling yellows isolates from mild and T36 genotype isolates. Using a dilution series of an in vitro synthesized transcript containing the target sequence, our protocol detected less than 2 fg of viral template. The broad spectrum assay was validated using total RNA extracts from fresh, frozen and desiccated leaf petioles from CTV-infected plants with 80 isolates of known biological activities from CTV collections from Beltsville, MD and Tulare and Parlier, CA. In the strain specific assay, 25 severe strains were distinguished from 22 mild CTV isolates from the different collections. These protocols are faster than ELISA and conventional RT-PCR and provides a new tool for detection and strain differentiation of CTV.