Development and validation of a real-time RT-PCR assay for generic detection of pospiviroids

In many countries phytosanitary regulations apply to Potato spindle tuber viroid, because it can cause serious diseases in potato and tomato crops. Other pospiviroids, some of which are distributed widely in ornamental crops, can cause similar diseases. Consequently, there is a need for a reliable and cost-effective generic testing method. An assay was developed that detects all known species of the genus Pospiviroid, using real-time RT-PCR based on TaqMan technology. This GenPospi assay consists of two reactions running in parallel, the first targeting all pospiviroids, except Columnea latent viroid, the second specifically targeting the latter viroid (already published). To monitor the RNA extraction a nad5 internal control was included. Method validation on tomato leaves showed that the GenPospi assay detects all pospiviroids up to a relative infection rate of 0.13% (equals 770 times dilution). The assay was specific because no cross reactivity was observed with other viroids, viruses or nucleic acid from plant hosts. Repeatability and reproducibility were 100% and the assay appeared robust in an inter-laboratory comparison. The GenPospi assay has been shown to be a suitable tool for large-scale screening for all known pospiviroids. Although it has been validated for tomato leaves it can potentially be used for any crop.