|Saponari,, Maria - INSTITUTE DI VIROLOGIA|
Submitted to: International Organization of Citrus Virologists Proceedings
Publication Type: Abstract Only
Publication Acceptance Date: August 20, 2007
Publication Date: October 17, 2007
Citation: Saponari,, M., Yokomi, R.K. 2007. Use of the CP and CPm Intergene Sequences to Discriminate CTV Strains. In: Proceedings of the 17th International Organization of Citrus Virologists, October 22-26, 2007, Adana, Turkey. p. 138. Technical Abstract: There is a need to develop a rapid assay to distinguish potentially mild vs severe strains of Citrus tristeza virus. Multiple alignment performed on the coat protein (CP) and the minor coat protein (CPm) intergene sequences (~80-100 bp) from different CTV isolates revealed that severe strains (VT, SY, T68 and T3 genotypes) generally associated with orange stem pitting (OSP), grapefruit stem pitting (GSP) and seedling yellows (SY) share a conserved sequence which is absent in mild isolates (T30-like genotype). Two assays were developed to differentiate such isolates: i) multiplex one-step real-time RT-PCR; ii) restriction fragment length polymorphism (RFLP) analysis. The real-time assay consisted of broad spectrum detection using a universal primers/Taqman probe (Cy5-labeled) and a primers/ MGB-Taqman probe (FAM-labeled) specific for severe genotypes. This assay allowed the simultaneous detection of all CTV isolates and differentiated between the mild and severe strains in our tests. The RFLP assay, however, was based on primers which amplified a target sequence containing unique restriction DdeI or SspI sites. The DdeI restriction site was conserved in severe and absent in mild strains. In contrast, the SspI restriction site was conserved in mild isolates and absent in the severe strains. Both assays were validated with a panel of known CTV isolates from the Exotic Citrus Pathogen Collection, USDA, ARS – Beltsville, MA and using natural or artificial combined infections. In conclusion, the two assays developed successfully differentiated between biologically different CTV strains.