engleski

Separation of plant viral satellite dsRNA using HPLC

Satellite RNAs are plant subviral agents which depend on their helper viruses for replication and spread. Those accompanied with cucumber mosaic virus are certainly most important in agricultural production inducing severe symptoms, particularly in tomato. Both virus and the satellite generate sufficient quantity of double stranded replictive RNAs (dsRNAs). These stable replicative forms can be isolated from small amount of infected tissue by CF-11 cellulose chromatography and recognized in standard electrophoretic profile. The approach is often used in preliminary identification of this extremely harmful pathogen combination. In our investigation this standard method was followed by high-performance liquid chromatography (HPLC) for further separation of the satellite dsRNA. Samples were run on Helix DVB Column (3x50 mm ; Varian) at linear gradient, 44-80% B in 12 minutes ; temperature 30°C, flow rate=0, 45 ml/min (A=0.1M TEAA (triethylammonium acetate), 0.1 mM EDTA, B=0.1M TEAA, 0.1mM EDTA, 25% ACN). Separation resulted in chromatogram showing strong peak from which the material was collected and the satellite dsRNA identified by electrophoresis and PCR. Once standardized, HPLC offer clear evidence of satellite presence, without precipitation and electrophoresis. At the same time it can be used as a preparative approach resulting in very pure satellite for further analyses.

cucumber mosaic virus(CMV); CMV satRNA; HPLC

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