Specific Detection of Cherry Mottle Leaf Virus Using Digoxigenin-Labeled cDNA Probes and RT-PCR

Abstract

Cherry mottle leaf virus (CMLV)-associated double-stranded RNA (dsRNA) was isolated from the propagation host Chenopodium quinoa. The dsRNA band, with a molecular weight estimated at 7.0 × 106 Da, was used to produce cDNA. Two recombinant plasmids from the cloned cDNA library were identified that specifically bound with CMLV-associated RNA in dot blot hybridization studies. The cDNA inserts were sequenced, and oligonucleotide primers were designed that specifically amplify an 848-bp fragment of the CMLV genome by reverse-transcription polymerase chain reaction. Also, a poly(T) primer was reliably used for reverse transcription, with specific amplification using the CMLV primers, suggesting polyadenylation of the virus genome. Search of the database revealed some sequence homology of the partially characterized genome of CMLV with that of apple chlorotic leafspot virus. Additional sequence data are required, however, to establish the taxonomic position of the filamentous CMLV.