Skip to main page content
Access keys NCBI Homepage MyNCBI Homepage Main Content Main Navigation
, 145 (2), 96-105

A Real-Time RT-PCR Assay for Detection and Absolute Quantitation of Citrus Tristeza Virus in Different Plant Tissues

Affiliations

A Real-Time RT-PCR Assay for Detection and Absolute Quantitation of Citrus Tristeza Virus in Different Plant Tissues

Susana Ruiz-Ruiz et al. J Virol Methods.

Abstract

A real-time RT-PCR assay using SYBR Green was developed for specific and reliable quantitative detection of Citrus tristeza virus (CTV) in infected plants. A general primer set designed from conserved sequences in ORFs 1b and 2 enabled amplification of the genomic RNA (gRNA) while excluding most subgenomic and defective RNAs. Single RT-PCR products of 204 bp (isolate T36) or 186 bp (other isolates) were obtained with no primer-dimer or non-specific amplifications detected. Melting curve analysis revealed distinct melting temperature peaks (T(m)) for severe and mild isolates. External standard curves using RNA transcripts of the selected target allowed a reproducible quantitative assay, with a wide dynamic range of detection starting with 10(2) gRNA copies and with very low variation coefficient values. This protocol enabled reliable assessments of CTV accumulation in different tissues and from different citrus species, grown in the greenhouse or under field conditions, and infected with CTV isolates differing in their pathogenicity. CTV accumulation was higher in bark and fruits than in roots or leaves and showed minimal differences among several susceptible citrus species, but it was significantly lower in sour orange. This quantitative detection assay will be a valuable tool for diagnosis and molecular studies on CTV biology.

Similar articles

See all similar articles

Cited by 10 PubMed Central articles

See all "Cited by" articles

Publication types

MeSH terms

LinkOut - more resources

Feedback