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

Abstract

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 (SCSMVAP), 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 μl and 10 μl/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 DACELISA tests, respectively. A cDNA clone pSCSMVAP (495 bp) specific to SCSMV-AP was selected as diagnostic ³²P and DIG (digoxigenin) probe. In slotblot hybridization analysis, ³²P 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.