Isolation of Double-stranded (ds) RNA from Virus-symptomatic Soybeans and Molecular Detection of Soybean mosaic virus (SMV) in North of Iran

Abstract

Background: There are a large number of pathogens which infect soybean crop. Viruses are one of these destructive agents. DsRNA extraction could be a reliable method to confirm the presence of a virus in a plant. To date, several attempts were conducted to extract dsRNA from different plants, but not from soybean.     

Objectives: As the symptoms of nutritional deficiency and viral diseases are very similar, intending to assess the presence of the virus (es), and particularly SMV, in soybean farms in the north of Iran, the dsRNA from soybean fresh leaf tissues were extracted.

Materials and Methods: In this study, the soybean samples were collected from the north of Iran followed by extraction of dsRNA using CF-11 column chromatography. DsRNA was run on 1% agarose gel and SMV was recognized by RT-PCR. The PCR product was bidirectionally sequenced.

Results: By using changes in other methods, we successfully extracted viral dsRNA from soybean. To confirm the dsRNA structure of the extracted molecules they were subjected to DNase I treatment; according to the results, dsRNA bands still existed after DNase I treatment which confirmed the RNA structure of the isolated molecules. According to the RT-PCR results, dsRNA extracted from SMV infected-soybean were confirmed through virus Helper Component Proteinase (HC-Pro) sequence amplification which verified the integrity of the extraction method.

Conclusions: In this study, it was demonstrated that dsRNA is a favorite tool for constructing of cDNA library, as it is more stable and resistant to RNase degradation in comparison with ssRNA, when the total RNA extraction is not feasible due to the low titer, instability of particles or the presence of inhibitors.