The development and application of a fast simultaneous detection to Solanaceae viroids

Viroids are circular non-coding single-stranded RNAs (249-401 nt) and could form secondary and tertiary structure by its special high complementary sequences. They are known as the smallest plant pathogen, and can infect wide variety of crops, including woody, herbaceous and horticultural plants. Until now, there are several viroids reported on the Solanaceae plants, including Potato spindle tuber viroid (PSTVd), Tomato chlorotic dwarf viroid (TCDVd), Pepper chat fruit viroid (PCFVd), Tomato apical stunt viroid (TASVd), Tomato planta macho viroid (TPMVd), Columnea latent viroid (CLVd). Though all the viroids above are not reported in Taiwan before, they have been listed as quarantine pests in the world. Thus, it is important and necessary to build up an efficient and quick diagnosis method to detect these viroids. This study has built a fast simultaneous detection for six Solanaceae viroids and the detection technique has been used on the import tomato seed viroid detection. Now, we have already detected 45 lots tomato seed from different countries including some of the Solanaceae viroid epidemic area. In addition, we also design several primer pairs of viroids for future multiplex RT-PCR, though these primer pairs still on test process, it would greatly reduce the amount of detection kit if the multiplex RT-PCR succeds. In this work, we have designed the primer pair CLVd-314 F/R, and combine the futher TASVd and TPMVd primer pairs to achieve three viroid multiplex PCR.