Studies on cDNA Probe Detection Technology for Apple Stem Pitting Virus in Kuala Pear

Using Kuala pear leaves and cortexes as materials, the total RNA was extracted using two methods and these two methods were compared. The most suited methods for Kuala pear were screened; and biotin-labeled cDNA probe was synthesized using RT-PCR. The main factors that affected the sensitivity of hybridization were studied. Studies indicated that the highest sensitivity was obtained under the following conditions: probe concentration 400 ng mL−1, formamide concentration 45%, temperature 42°C, hybridization time 6 hours. The best hybridization results were obtained when the nitrocellulose membrane purchased from Gelman was used. Better blocking of hybridization was obtained using Tween 20 compared with albumin in bovine. The detection of the total RNA using different tissues and different extraction methods was compared. This study indicates that the total RNA of fresh leaf, old leaf, cortexes, and frozen leaf showed signs of hybridization using the two extraction methods.