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.
Potato blackleg caused by Dickeya spp. bacteria is one of the most important bacterial diseases of potatoes. The rapid spread of this disease in the territory of Russia requires new effective diagnostic tools for the timely detection of infection. To solve this problem, antisera specific to Dickeya spp. were obtained. Polyclonal antibodies isolated from antisera have shown high affinity for the main species of Dickeya spp. ( D. solani, D. dianthicola, D. chrysanthemi, D. dadantii, D. paradisiaca ). Enzyme linked immunosorbent assay (ELISA) and lateral flow immunoassay (LFIA) test systems have been developed based on specific and high affinity antibodies that were obtained. For ELISA, the detection limit was 0.8 105 cells/mL for D. solani and 2 104 cells/mL for D. dianthicola . For LFIA, suitable for use in non-laboratory conditions, the detection limit of D. solani was 2 105 cells/mL and the analysis time was 15 minutes. When testing potato seed material, LFIA test system confirmed positive results of ELISA determination in 75 % of samples, and negative - in 100 % of samples.
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