DsRNA Cloning and Diagnosis of Beet Pseudo-Yellows Virus by PCR and Nucleic Acid Hybridization

Abstract

DsRNA has been extracted from beet pseudo-yellows virus infected cucumber plants, purified to homogeneity, and cDNA clones to it produced. The clones are of insufficient sensitivity to detect infection-specific RNA in dot and northern blots of crude nucleic acid extracts. However, a knowledge of the sequence of these clones has been used to synthesize oligonucleotides that have been used for polymerase chain reaction amplification of specific sequences from both purified dsRNA and from infected plants and used as a previously unavailable highly sensitive diagnostic probe. The method of cDNA synthesis has been shown to be generally applicable to some other plant viral dsRNAs and should be of use in the production of cDNA clones when virion RNA is unavailable.

Screening of the closterovirus genome by degenerate primer-mediated polymerase chain reaction

“…1 a). The largest band of BPYV corresponded to a fragment of approximately 9 kbp, in agreement with previously published estimates (Coffin & Coutts, 1992), and the largest LIYV dsRNA was estimated to be around 12 kb (Fig. 1).…”

“…Lettuce infectious yellows virus (LIYV) induces formation of BYV-type vesicles in the infected cells, but is transmitted by whiteflies (Duffus et al, 1986). The whiteflytransmitted beet pseudo-yellows virus (BPYV) does not induce formation of BYV-type vesicles and has a genome of only about 9 kb (Liu & Duffus, 1990;Coffin & Coutts, 1992). So far the genetic similarities between these viruses are unclear.…”

Cherry virus A: cDNA cloning of dsRNA, nucleotide sequence analysis and serology reveal a new plant capillovirus in sweet cherry

“…DsRNA templates have been used for eDNA cloning as an alternative to genomic RNA template (Pappu et al, 1994), particularly when virion RNA is unavailable (Coffin & Coutts, 1992;Jelkmann et al, 1992b;Jelkmann, 1994). Pappu et al (1994) obtained nearly identical results by nucleotide sequencing two sets of eDNA clones generated from genomic RNA and dsRNA of a large portion of citrus tristeza closterovirus.…”

“…Extraction of dsRNA from plant tissue infected with viruses with ssRNA genomes and the subsequent use of this nucleic acid template for cDNA cloning has been demonstrated to be a useful alternative to the use of ssRNA templates when virion RNA is unavailable (Jelkmann et at., 1989;Coffin & Coutts, 1992). This method has been used to obtain partial (Pappu et al, 1994) or complete virus genomes (Jelkmann et al, 1992b;Jelkmann, 1994) of recalcitrant fruit viruses.…”

Incidence of Viruses Infecting Cucurbits in Cyprus

“…Reverse transcription for ZYMV and CVYV was performed using total RNA from infected plants, as described by Coffin and Coutts (1992). It was performed at 42°C for 60 min, in a 10 μ l reaction volume using 2 μ l of total RNA, 80 units M‐MLV reverse transcriptase (Invitrogen, Breda, the Netherlands) according to manufacturer's recommendations, 1 μ m oligo‐(dT) 18 primer and 20 units ribonuclease inhibitor (HT Biotechnology, Cambridge, England).…”