Development of a Test-Systems for the Identification of the Soil Borned Tobacco Rattle Virus Based on the Use of Elisa

Abstract

On the base of the isolate TRV PV-0361 of the tobacco rattle virus from the commercial collection of the DSMZ company (Germany) have been developed methods for maintaining the virus in vitro culture on N. clevelandii plants, propagation the virus in the spring-summer period in greenhouse conditions, “soft” isolation of a purified virus preparation by clarifying leaf juice with low-speed centrifugation and treatment with non-ionic detergent Triton-X-100 followed by precipitation of the virus with polyethylene glycol-6000 and three-fold ultracentrifugation using sucrose cushion, sucrose concentration gradient and differential centrifugation. The purified virus preparation was used for producing rabbit antiserums according to the scheme we worked out. The obtained antiserum had the following titers - specific 1: 5 · 105, non-specific 1: 8 · 103. Based on antibodies isolated from this antiserum, coating antibodies and peroxidase conjugates were obtained, which made it possible to create ELISA test systems for determining TRV with sensitivity of about 12-16 ng/ml. The resulting test systems can be used in practical work on quality control and certification of seed potatoes.