Diagnosis of Flame Chlorosis by Reverse Transcription-Polymerase Chain Reaction (RT-PCR). S. Haber, Agriculture and Agri-Food Canada, Research Centre, Winnipeg, MB R3T 2M9. J. D. Procunier, Agriculture and Agri-Food Canada, Research Centre, Winnipeg, MB R3T 2M9, G. Murray, Department of Microbiology, University of Manitoba, Winnipeg MB R3T 2N2, and S. E. Cvitkovitch, Agriculture and Agri-Food Canada, Research Centre, Winnipeg, MB R3T 2M9. Plant Dis. 79:626-630. Accepted for publication 14 February 1995. Copyright 1995 Department of Agriculture and Agri-Food, Government of Canada. DOI: 10.1094/PD-79-0626.

Flame chlorosis (FC), a viruslike disease of cereals, is associated specifically with double-stranded RNAs (FCdsRNAs). The FCdsRNAs can be detected by dot blot hybridization assay that is adequate for detecting FC-RNA in symptomatic areas of infected leaf tissue, but has proved insufficiently sensitive to detect FC-RNA in small (?10 mg) quantities of suspect root tissue or mycelium of candidate fungal vectors. A reverse transcription-polymerase chain reaction assay to detect FC-RNA (FC-RT-PCR) was developed to improve sensitivity. Total RNA was extracted from milligram quantities of test tissue, reverse transcribed, and amplified by the PCR. The primer pairs #86 [F:5'-CTATTCGCTTGGCTCAGATCG-3' and R:5'-CCAGAGTA-GTGACTAGAACAGC-3'] or #307 [F:5'-GTGAAAGTCTTGAGGATGC-3' and R:5'-TTCA-TCTCTATTGGCACCACG-3'] were used. These primer pairs had been determined from a consensus 821-bp FC sequence covering an open reading frame (GenBank No. X59248), and were predicted to yield 358- and 347-bp DNA fragments, respectively. Sensitivity of specific FC-RNA detection was further enhanced by hybridization of the RT-PCR product to digoxi-genin-labeled riboprobe (digFC-RNA) used in the earlier FC dot blot hybridization assay; the digoxigenin label was subsequently reported with enzyme-linked antidigoxigenin antibody and chromogenic substrate

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