Broad-spectrum detection and quantitation methods of Soil-borne cereal mosaic virus isolates

Abstract

A broad-spectrum reverse transcription-polymerase chain reaction (RT-PCR) protocol was developed for detecting Soil-borne cereal mosaic virus (SBCMV) isolates, responsible for mosaic diseases in Europe, using primers targeting the highly conserved 3′-untranslated region of RNA-1 and RNA-2 of SBCMV. The 3′-end region is a privileged target for the detection of a wide range of isolates, because of sequence conservation, of the tRNA-like structure, the major role in viral replication and the signal amplification due to the presence of numerous genomic and subgenomic RNAs. The primers were also designed for virus quantitation using real-time RT-PCR with SYBR-Green chemistry. No cross-reaction with Wheat spindle streak mosaic virus, frequently associated with SBCMV, was observed. The use of RT-PCR and real-time quantitative RT-PCR allowed a more sensitive detection and quantitation of SBCMV to be made than was the case with ELISA. The methods enabled European isolates of SBCMV from Belgium, France, Germany, Italy and the UK to be detected and quantified. Real-time RT-PCR represents a new tool for comparing soil inoculum potential as well as cultivar resistance to SBCMV.