Detection of Apple scar skin viroid in apple trees by the isothermal nucleic acid amplification and electrochemiluminescence

A rapid and sensitive assay for the specific detection of Apple scar skin viroid (ASSVd) using nucleic acid sequence-based amplification (NASBA) combined with electro-chemiluminescence (ECL) detection was established. Nucleic acids were isolated from symptomless young leaf discs of ASSVd-infected apple tree by a silicabased method. For this purpose we designed a set of ASSVd-specific primers to allow amplification of viroid RNA, and a biotin-labelled specific capture probe complementary to an internal region of the viroid. Following a properly optimized NASBA step, the amplified ASSVd RNA was detected by ECL after hybridization to the capture specific probe. The NASBA-ECL method was faster and 100-fold more sensitive than RT-PCR for ASSVd detection in apple leaves. ASSVd was also reliably detected in bark tissue from infected apple trees when polyvinylpyrrolidone was included in the extraction buffer to sequester the polysaccharides which, otherwise, inhibited the reaction. This is the first report on the successful application of a NASBA-ECL technique for the detection of a viroid pathogen.