Evaluation of viroid extraction methods and application of a one-step reverse transcription real-time polymerase chain reaction assay (RT-qPCR) for the rapid detection of Chrysanthemum stunt viroid (CSVd) infection

Early viroid detection in chrysanthemum cultivars depends on both an efficient RNA extraction method as well as a sensitive detection assay. In this study, we evaluated RNA purification methods and optimized a one-step RT-qPCR assay for the detection of Chrysanthemum stunt viroid (CSVd). Three commercially available RNA extraction technologies (silica and silicon carbide spin column as well as the phenol/chloroform extraction) were compared for efficacy in CSVd detection sensitivity. We found a 10- to 50-fold improvement in CSVd detection sensitivity when using silicon carbide (SiC) spin columns to purify RNA compared with the silica-based column or the traditional phenol/chloroform extraction method. To optimize detection sensitivity and reduce detection time, a commercial CSVd end-point PCR primer set was adapted to a SYBR Green based one-step RT-qPCR assay. Standard curve analysis estimated that the limit of detection was about 54 CSVd copies and the specificity test against six related viroids showed no cross-reactivity. As a part of the validation, the one-step RT-qPCR detection system was compared with RT-PCR to detect CSVd in both randomly selected greenhouse plants and mechanically inoculated chrysanthemum varieties. Cultivars selected from a commercial greenhouse with > 50% infection levels were tested for CSVd presence. Cultivars 'Pelee', 'Puma' and 'Shamrock' tested positive by both the one step RT-qPCR and RT-PCR but CSVd was not detected in 'Icecap' and 'Snowball'. In mechanical inoculation studies, CSVd was detected in 5 of 6 chrysanthemum cultivars - 'Chesapeake', 'Durango', 'Juneau', 'Pelee' and 'Viron' but not in 'Pueblo'. No differences in detection were evident between the RT-qPCR and RT-PCR methods.