A two-step multiplex RT-PCR method for simultaneous detection ofsoil-borne wheat mosaic virus and wheat spindle streak mosaic virus fromFrance

摘要

A sequence of 942 nucleotides, located in the helicase gene of RNA1 of a French isolate of soil-borne wheat mosaic virus (SBWMV), is presented. This sequence was compared with the corresponding sequences published for Nebraskan and Chinese isolates and showed a 74.8% and 73.4% identity, respectively, with these isolates, whereas the Nebraskan and Chinese isolates shared a 78.2% identity. A set of primers specific to the French SBWMV isolate was designed on the basis of this sequenced 942 nucleotide fragment. A primer set for wheat spindle streak mosaic virus (WSSMV) was designed from the published partial sequence of a French isolate. Both sets of primers were combined into a two-step multiplex reverse-transcription polymerase chain reaction, allowing simultaneous detection of both viruses in leaves of infected wheat samples. The amplification specificity of the two sets of primers was checked against isolates of SBWMV from Oklahoma and China, Indian peanut clump virus strains H, L and D from India, barley mild mosaic virus and barley yellow mosaic virus from France. SBWMV primers were specific for French isolates of this virus, whereas the primers designed from the sequence of a French isolate of WSSMV could also faintly detect barley yellow mosaic virus in barley plants. The RT-PCR technique was also compared with ELISA (enzyme-linked immunosorbent assay) on 100 wheat leaf samples collected from the field on the basis of symptoms, and was shown to be reliable and reproducible.