|Saponari,, Maria - ISITUTO DI. VIROL VEG.|
Submitted to: Journal of Plant Pathology
Publication Type: Abstract Only
Publication Acceptance Date: August 12, 2007
Publication Date: December 1, 2007
Citation: Saponari,, M., Yokomi, R.K. 2007. Real-time RT-PCR assay for detection and differentiation of Citrus tristeza virus isolates. Journal of Plant Pathology 89 (3, supplement):S60. Technical Abstract: For universal detection of Citrus tristeza virus (CTV) strains by real time RT-PCR, a protocol was developed based on a set of primers and a Cy5-labeled TaqMan probe. This test included primers and a TET-labeled TaqMan probe selected on the mitochondrial nad5 gene for the simultaneous detection of host RNA as an internal control. This protocol was tested on total RNA extracts from fresh, frozen, and desiccated leaf petioles from CTV-infected plants with 80 isolates from a worldwide collection of CTV isolates maintained in planta in Beltsville, MD and Tulare and Parlier, CA, USA. A dilution series of an in vitro synthesized transcript containing the target sequence showed the protocol detected less than 2 fg of viral template. A multiplex, one-step real-time RT-PCR assay was also developed for the detection and differentiation of CTV strains. Based on multiple alignments of the nucleotide sequences of the CTV minor and major coat proteins, a set of primers and FAM-labeled TaqMan probe were developed to detect stem pitting and seedling yellows CTV strains (VT and T3 genotypes). This assay was accurate when the two primers and TaqMan sets were combined and allowed the simultaneous detection and differentiation of mild and severe strains of the virus. This test successfully distinguished all 25 severe from 22 mild isolates from an ad hoc group of CTV isolates.