Development of cDNA probe for the detection of rice ragged stunt virus
1995
Arpornpun Pochanukul
Rice ragged stunt virus (RRSV) causes a serious disease in rice and is transmitted in a persistent manner by brown planthoppers. The disease is sporadic and widely distributed in the Tropical Asian countries including Thailand. The genome of RRSV comprises 10 segments of double-stranded RNA (dsRNA). The dsRNA was extracted from RRSV infected rice tissue by using CF-11 column and further purified through agarose gel electrophoresis. The dsRNA was eluted from agarose gel by Geneclean. The cloning of dsRNA which was purified through agarose gel and denatured by boiling at 96 deg C for 8 min was accomplished. After first and second strand synthesis, the DNA was size fractionated on a Sephacryl S-400 column, EcoRI adaptor-tailed, annealed to EcoRI-cut pUC 118 plasmid vector and transformed to Escherichia coli JPA 101 cells by electroporation. Ten of 406 white colonies randomly picked were found to have recombinant plasmid contained inserts range from 600 to 3000 basepairs. Clones named R102, R105, R106 and R108 which contained inserts of 1700, 800, 3000 and 1400 basepairs respectively were used as templates for preparation of nonradioactive cDNA probe with digoxigenin dUTP label. cDNA probe obtained from R105 clone was efficient to detect dsRNA from infected rice and weed host at the level as low as 1 picogram. The cDNA probe was further applied for virus detection in weed hosts collected from paddy field. In 1994, weeds collected from Nakhon Pathom, Pathum thani and Suphan Buri gave negative results for RRSV detection.
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