Cloned cDNA probes for the detection of tomato spotted wilt virus
1989
Ronco, A.E. | Dal Bo, E. | Ghiringhelli, P.D. | Medrano, C. | Romanowski, V. | Sarachu, A.N. | Grau, O.
As a first step toward the development of a detection test for tomato spotted wilt virus (TSWV) based on nucleic acid hybridization, cDNA clones have been obtained and used to probe viral RNA from purified virions and infected plants. DNA reverse transcribed from TSWV unfractionated RNA was ligated to linearized pUC13 plasmid and cloned in Eicherichia coli. Cloned inserts ranged from 0.13 to 1.0 kb and represented TSWV RNA2 and RNA3 sequences. Two clones--0.73 and 1.03 kb long--were labeled with 32P by nick translation and used as probes in dot blot and Northern blot hybridization assays. The dot blot assay allowed detection of as little as 2 ng of RNA from purified virions as well as TSWV RNA present in total RNA extractions from 80 mg of infected leaves of Nicotiana rustica and in crude saps from 8 mg of the same plants. After a slight modification in the homogenization procedure, TSWV RNA was detected in crude extracts from 3 mg of infected tomato plants, by the dot blot assay. Viral RNA was also unambiguously detected in total RNA preparations from 5 g of infected tomato leaves by Northern blot hybridization.
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