A two‐step multiplex RT‐PCR method for simultaneous detection of soil‐borne wheat mosaic virus and wheat spindle streak mosaic virus from France

Abstract

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.

Response of UK winter wheat cultivars to Soil-borne cereal mosaic and Wheat spindle streak mosaic viruses across Europe.

“…SBCMV has been found subsequently at 12 further sites in the UK, including the Isle of Wight, Kent and Northampton. In Europe, WSSMV was first detected in France in 1977 (Signoret et al 1977), and is now known to occur throughout Belgium, France, Germany and Italy (Gitton et al 1999;Huth 2000;Rubies Autonell and Vallega 1987;Vaianopoulos et al 2006). WSSMV is often found infecting wheat in combination with SBCMV, but this virus has not yet been reported in the UK.…”

Multiplex Reverse Transcription-PCR for Simultaneous Detection of Beet Necrotic Yellow Vein Virus , Beet Soilborne Virus , and Beet Virus Q and Their Vector Polymyxa betae KESKIN on Sugar Beet

“…It has already been applied with success to the detection of poleroviruses infecting sugar beet (11) as well as soilborne viruses of potato (27) and cereals (10). This article describes an mRT-PCR for the simultaneous detection of BNYVV, BSBV, BVQ, and P. betae.…”

“…mRT-PCR tests have already been proposed to detect plasmodiophoromycete-transmitted viruses like Potato mop top virus (27), Soilborne wheat mosaic virus, and Wheat spindle streak mosaic virus (10). The proposed mRT-PCR technique allows the simultaneous detection of BNYVV, BSBV, BVQ, and their vector P. betae.…”

Molecular Techniques for Detection of Microbial Pathogens

“…Amplification of two or more specific DNA fragments simultaneously can be done by using a combination of several primers in the same PCR assay. Such multiplex RT-PCR methods have been applied to detect multiple species of a virus in a single plant as in the case of detection of Cymbidium mosaic virus (CyMV) and Odontoglossum ringspot virus in orchids (Seoh et al 1998), Soilborne wheat mosaic virus and Wheat spindle streak mosaic virus (Gitton et al 1999), three ilarviruses affecting stone fruit trees (Saade et al 2000), six citrus viroids and Apple stem grooving virus from citrus (Ito et al 2002) and Criniviruses causing yellowing disease in tomatoes in Greece (Dovas et al 2002). A multiplex RT-PCR, using a cocktail of nine primers was developed for the detection of five seedborne legume viruses by Bariana et al (1994).…”