Selection of PCR primers and a simple extraction method for detection of hop stunt viroid-plum in plum reverse transcription-polymerase chain reaction

• Published in: Nippon shokubutsu byōri gakkai Vol. 63; no. 2
• Main Authors: Kusano, N. (Fukuoka-ken. Agricultural Research Center, Chikushino (Japan)), Shimomura, K
• Format: Journal Article
• Language: Japanese
• Published: 1997
• Subjects: ENFERMEDADES DE LAS PLANTAS; MALADIE DES PLANTES; PCR; PLANT DISEASES; PRUNUS SALICINA; VIROIDE; VIROIDES; VIROIDS
• ISSN: 0031-9473
• DOI: 10.3186/jjphytopath.63.119

Several conditions were examined to optimize the detection of hop stunt viroid-plum, a causal pathogen of plum dapple fruit disease, by reverse transcription-polymerase chain reaction (RT-PCR). The best primer pair of the six examined was HSVc-1(5'-GGCTCCTTTCTCAGGTAAG-3')/HSVs-2 (5'-CCGGGGCAACTCTTCTCAGAATCCA-3'). By using this primer pair, the RT-PCR assay was about 10,000 times more sensitive than two-dimensional polyacrylamide gel electrophoresis, judging from a dilution end point experiment. Treatment of CF-11 cellulose column chromatography before RT-PCR was effective in removing inhibitors present in plum sap extracts. SepaGene RV-R was satisfactory as a simple extrication method to extract the viroid from barks, roots and leaves of plum for RT-PCR assays. The extracts from barks and roots gave better PCR amplification than leaf extracts. The direct nucleotide sequencing of PCR-amplified cDNAs of the viroid isolated from dapple fruit plum (cvs. Ooishiwasesumomo and Soldam) revealed that the viroid was identical to HSVd-plum reported in plum (cv. Taiyo) in Yamanashi prefecture