Dot Blot Detection of Tomato Spotted Wilt Virus RNA in Plant and Thrips Tissues by cDNA Clones

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Abstract
A cDNA library was constructed that contained sequences complementary to tomato spotted wilt virus (TSWV) RNA. Two plasmids, pTSWV7 and pTSWV80, containing inserts of 460 and 870 bp, respectively, were characterized for specificity to TSWV RNA and were selected to develop a diagnostic dot blot assay. 32P-labeled hybridization probes from these clones can detect TSWV in as little as 16 ng of total RNA from tobacco, 80 ng from tomato and lettuce, and 400 ng from chrysanthemum and pepper. TSWV is detected by these hybridization probes in dot blots of nucleic acids extracted by simple methods using 40-50 mg of leaf tissue and in all the major developmental stages of individual thrips.