Isolation and Analysis of Double-Stranded RNA from Virus-Infected Plant and Fungal Tissue

TLDR

The study presents a simple, rapid method to isolate dsRNA from virus‑infected plant and fungal tissues for virus detection and identification. The method uses phenol extraction of diseased tissue followed by selective purification of dsRNA on cellulose powder in 15 % ethanol, then analysis by gel electrophoresis and ribonuclease treatment, enabling rapid isolation from small tissue amounts. Successful isolation of dsRNA does not depend on tissue type, making the method useful for studying RNA virus replication and diagnosing infections directly from host tissues.

Abstract

A simple, rapid method for the isolation of double-stranded RNA (dsRNA) from virus-infected plant and fungal tissues provides a new approach to virus detection and identification. Diseased tissue was phenol-extracted to isolate cellular nucleic acids, and viral dsRNA was selectively purified from other nucleic acids by binding to cellulose powder in 15% ethanol either in small columns or by a batch procedure. The product was analyzed first by gel electrophoresis and then by ribonuclease treatment to identify dsRNA. The method permits rapid and efficient isolation and analysis of dsRNA from small amounts (1-10 g) of tissue and from multiple samples using small amounts (0.1-2.5 g) of cellulose powder. Successful isolation of dsRNA does not depend on the type of tissue processed and the method therefore is potentially useful for the study of RNA virus replication and for detection and diagnosis of virus infections directly from the infected host tissues.