Development of a comprehensive detection and identification molecular based system for eight pospiviroids

Pospiviroids cause serious economic damage to solanaceous crops, including tomato (Solanum lycopersicum), and cause symptomless infections in many horticultural plants. Symptomless infection and seed transmission are obstacles in visual inspection for plant quarantine. Therefore, in the present study, a comprehensive detection and identification system for molecular diagnosis of eight pospiviroids was developed, based on real-time reverse transcription polymerase chain reaction (RT-PCR). In this system, two real-time RT-PCR methods based on SYBR Green and TaqMan technologies were combined. First, tomato seed samples were screened for all the eight pospiviroids by the SYBR Green method using three primer pairs, including one pair of universal primers. Samples that were positive using the universal primer set were subjected to secondary screening for the identification to species level of six viroids by TaqMan real-time RT-PCR method, with specific primer and probe sets. This two-phase system could specifically detect a viroid in a sample of 400 seeds containing a single contaminated seed. The developed system could be an effective and simple tool to avoid the introduction of pospiviroids through tomato seeds.