A novel RT-PCR approach for detection and characterization of citrus viroids

Citrus plants are natural hosts of five viroid species and a large number of sequence variants. Because of their small size, viroids lend themselves to various RT-PCR approaches for their detection and further characterization. The one-step RT-PCR approach proposed here is based on the synthesis of viroid-cDNA by reverse transcription at 60 degrees C using a viroid specific 27-mer primer followed by standard second strand synthesis plus PCR amplification with various primer pairs. According to the primers used, full or partial length viroid-DNA is obtained. The technique avoids amplicon contamination in routine diagnosis. The suitability of the technique has been demonstrated using several nucleic acid extraction procedures and different viroid infected host species. The homogenization of tissue inside sealed plastic bags followed by nucleic acid extraction using a SDS/potassium acetate method is recommended because of its efficiency, simplicity and low cost. This extraction procedure, when coupled to the one-step RT-PCR approach, can be useful to avoid cross-contamination during routine diagnosis. A PCR strategy capable of discriminating between mild and severe strains of CEVd and identifying cachexia-inducing isolates of HSVd, is also described.