Real-Time RT-PCR Based on SYBR-Green I for the Detection of Citrus Exocortis and Citrus Cachexia Diseases

Abstract

Citrus exocortis viroid (CEVd) and Citrus viroid-IIb (CVd-IIb) are the etiological agents of citrus exocortis and citrus cachexia diseases, which cause economically important damage in citrus crops. For this reason they are both included in citrus certification programs. The need to speed up the detection of pathogens has stimulated progressive improvement in and application of appropriate technology. In the present study we have devised real-time RT-PCR protocols using the intercalating dye SYBR-Green I for fast detection of these two pathogens in field samples infected with a mixture of viroids. Primer pairs designed from highly conserved regions of the genome of different variants of each viroid amplified DNA fragments of 83-bp (CEVd) and 133-bp (CVd-II), which were detected by the increasing fluorescence observed during the reaction. The analysis of the melting temperatures (Tm) allowed for the discrimination of the amplified products generated from the two distinct viroid templates. The presence of products specific to CEVd, cachexia and non-cachexia CVd-II was always confirmed by visualization with polyacrylamide gel electrophoresis. The evidence indicates that real-time RT-PCR appears to be a useful tool for fast and reliable diagnosis of citrus viroids.