Publications - Faculty - Recently Submitted Faculty Publications 2009
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Brlansky - Journal of Virological Methods
Characterization of the mixture of genotypes of a Citrus tristeza virus isolate by quantitative PCR assays
G. Ananthakrishnan, T. Venkataprasanna, A. Roy and R. H. Brlansky
Abstract - A real-time multiplex RT-PCR assay using Taqman® RNA-to-CtTM one-step kit was developed to detect and quantify the genotypic mixture present in virus infected plants. Citrus tristeza virus (CTV) isolate FS627, a complex Florida isolate containing T36, T30 and VT genomes and its aphid transmitted subisolates were used. The gene expression assay was carried out using specific primers and probes developed from the genomes of three CTV virus isolates and included the coat protein region of isolate T36 and the 5′ end, ORF 1a and ORF 2 region of isolates T36, T30 and VT. Among the three genomes present in the aphid transmitted subisolates, the T30 genome showed higher overall gene expression in all specific regions compared to other isolates. The profiles of the some subisolates were different from the parent source from which they transmitted. The 2-ΔΔCt method was used to analyze the relative titers of the three reference genomes in the aphid transmitted plants infected with FS627. This protocol enabled assessments of CTV genetic diversity in the aphid transmitted subisolates. This simple quantitative assay was sensitive, efficient and took less time than other existing methods. This relative quantitative assay will be a reliable tool for diagnosis, detection and gene expression studies on CTV.
