Abstract:

In order to simplify the conventional RT-PCR of apple skin scar viroid and find faster and more accurate detection methods, the apple shoots in field infected by apple skin scar viroid were used as materials to optimize the RT-PCR systems. Specific primers of apple skin scar viroid were designed and synthesized according to the gene sequences in Genebank and domestic kit was selected in this study. The results showed that under the conditions of the amount of total RNA 37.4-74.8 ng, reverse transcription products 1.0-3.0 μL and the annealing temperature 63.8℃, two-step RT-PCR system got a good amplification. While the amount of total RNA 3.74-7.48 ng and the annealing temperature from 50.1 to 62.6℃, the detection systems of one-step RT-PCR got the better amplification. The two RT-PCR detection systems were tested with samples collected from Shandong Province, and the results were completely consistent. This study established the quick and reliable detection method of apple scar skin viroid by the two-step and the one-step RT-PCR by kit.