Antibody and nucleic acid probe-based techniques for detection ofsugarcane streak mosaic virus causing mosaic disease of sugarcane in India

摘要

Double antibody sandwich-enzyme linked immunosorbent assay (DAS-ELISA) and direct antigen coating (DAC)-ELISA tests were evaluated for detection of sugarcane streak mosaic virus (SCSMV-AP), a new member of Tritimovirus genus in the family Potyviridae, in leaf extracts, sugarcane juice and purified virus. The virus was detected up to 1/3125 and 1/625 dilutions in infected sugarcane leaf, 5 mul and 10 mul/well in sugarcane juice, 1/3125 and 1/3125 dilutions in infected sorghum leaf and 10 ng and 50 ng/ml for purified virus in DAS-ELISA and DAC-ELISA tests, respectively. A cDNA clone pSCSMV-AP (495 bp) specific to SCSMV-AP was selected as diagnostic P-32 and DIG (digoxigenin) probe. In slot-blot hybridization analysis, P-32 and DIG probes reacted with total nucleic acid extracts from infected sugarcane leaf (10(-5) and 10(-4)), infected sugarcane juice (10(-3) and 10(-4)) and infected sorghum leaf (10(-5) and 10(-5)). With both the probes the virus was detected in purified virus up to 50 pg and viral RNA up to 1 pg level. DAS-ELISA appears to be sensitive and ideal for routine large-scale detection of virus in small leaf tissue and cane juice samples of sugarcane. Nucleic acid-based tests could be useful in screening of sugarcane germplasm.