Duplex RT-PCR assay for simultaneous detection of TSWV and CSVd in chrysanthemum.

A novel duplex RT-PCR assay for simultaneous detection of TSWV and CSVd in chrysanthemums was developed. Previous reported primers for amplification of TSWV and CSVd were used and a novel pair of primers for CSVd was designed to improve duplex amplification compatibility. Sensitivity and efficiency of the previous reported and novel primers for CSVd were assessed. Then, the sensitivity of the combined primers to amplify both TSWV and CSVd cDNA were also evaluated. Both TSWV and CSVd were detected in preparations diluted up to 10-4 and 10-5 respectively, from total RNA extracts. This duplex RT-PCR method showed an estimated diagnostic sensitivity (DSe) of 97% and diagnostic specificity (DSp) of 99%. For combination of the primers TSWV L1/ L2 and CSVd UCO-1 F/ UCO-1R, the protocol could detect pathogen RNA from naturally infected plants until 0.1 ng and 1 ng respectively. This novel protocol for detection of TSWV/CSVd represents a useful diagnostic tool without the need of expensive probes and less extensive laboratory work. This method could be helpful to assist the selection and further propagation of healthy chrysanthemums on the field as well as to understand the dynamics and the interaction of this virus and viroid within farms.