DIAGNOSIS OF FLAME CHLOROSIS BY REVERSE TRANSCRIPTION-POLYMERASE CHAIN-REACTION (RT-PCR)

Flame chlorosis (FC), a viruslike disease of cereals, is associated sp ecifically with double-stranded RNAs (FCdsRNAs). The FCdsRNAs can be d etected by dot blot hybridization assay that is adequate for detecting FC-RNA in symptomatic areas of infected leaf tissue, but has proved i nsufficiently sensitive to detect FC-RNA in small (less than or equal to 10 mg) quantities of suspect root tissue or mycelium of candidate f ungal vectors. A reverse transcription-polymerase chain reaction assay to detect FC-RNA (FC-RT-PCR) was developed to improve sensitivity. To tal RNA was extracted from milligram quantities of test tissue, revers e transcribed, and amplified by the PCR. The primer pairs #86 [F:5'-CT ATTCGCTTGGCTCAGATCG-3' and R:5'-CCAGAGTAGTGACTAGAACAGC-3'] or #307 [F: 5'-GTGAAAGTCTTGAGGATGC-3' and R:5'-TTCATCTCTATTGGCACCACG-3'] were used . These primer pairs had been determined from a consensus 821-bp FC se quence covering an open reading frame (GenBank No. X59248), and were p redicted to yield 358- and 347-bp DNA fragments, respectively. Sensiti vity of specific FC-RNA detection was further enhanced by hybridizatio n of the RT-PCR product to digoxigenin-labeled riboprobe (digFC-RNA) u sed in the earlier FC dot blot hybridization assay; the digoxigenin la bel was subsequently reported with enzyme-linked antidigoxigenin antib ody and chromogenic substrate.