Diagnosis of Specific Viral RNA Sequences in Plant Extracts by Hybridization with a Polynucleotide Kinase-Mediated,32P-Labeled, Double-Stranded RNA Probe

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Abstract
A method was developed for screening of virus-related RNA in plant extracts. The method is based on the hybridization of a polynucleotide kinase-mediated, 32P-labeled, double-stranded (ds) RNA probe with RNA or sap extracts. The cucumber mosaic virus (CMV) and associated RNA-5(CARNA-5) was used as a model system. Naturally infected plants of Nicotiana glauca were screened for the presence of dsCARNA-5; dsRNA was purified on CF-11 columns and analyzed in polyacrylamide gels. Three types of low-MW CARNA-5-like dsRNA species, with estimated MW of (type 1) 0.27, (type II) 0.22 and (type III) 0.19 .times. 106 daltons, were observed in plants of N. glauca that reacted with CMV antiserum. Types II and III contained CARNA-5 sequences by Northern blot hybridization of dsRNA patterns with 32P-labeled dsCARNA-5. This labeled probe was further used to detect CARNA-5 infection in N. glauca and N. glutinosa plants by hybridization with dot-spots of dsRNA as well as plant saps. The applicability of 32P-labeled dsRNA for diagnosing specific sequences of plant virus RNA is discussed.