Details

Autor(en) / Beteiligte

• Tessitori, M
• Rizza, S
• Reina, A
• La Rosa, R

Titel

Development of a real-time PCR assay for the simultaneous detection of citrus viroids

Ist Teil von

• Journal of plant pathology, 2004-12, Vol.86 (4), p.336-336

Erscheinungsjahr

2004

Beschreibungen/Notizen

• Five distinct viroid species with about 20 sequence variants affect citrus species. Usually, field isolates are often composed by a mixture of two or more viroids. CEVd (Citrus exocortis viroid) and CVd-IIb (Citrus viroid-IIb) are widespread in the main citrus areas causing two economically important diseases, exocortis and cachexia. In order to meet citrus certification program requirements the search for a rapid and efficient diagnostic method to detect citrus viroids is still in progress. A protocol of SYBR-Green I real-time RT-PCR was set up for CEVd and CVd-II detection as an evolution of classical RT-PCR. Efficient real-time primer pairs were designed for each viroid in highly conserved regions of the genome and the protocol was tested on field samples containing a mixture of both viroids. The specificity of the reaction was confirmed by the melting temperatures (Tm), which were consistently specific for both amplicons obtained. All protocols were developed amplifying the cDNA obtained from each viroid using OmniMix beads hot start master mix (TaKaRa Bio Inc.). Advantages of real-time PCR technique coupled with use of Smart-Cycler TDII System (Cepheid Europe SA), a real-time apparatus that can perform 16 independent reactions, allowed developing a sensitive and rapid method for the simultaneous detection of both viroids.