Submitted to: Journal of Virological Methods
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 10/12/2007
Publication Date: 12/1/2007
Citation: Mahua, D., Anderson, J.M. 2008. Development of a multiplexed PCR detection method for Barley and Cereal Yellow Dwarf viruses, Wheat Spindle Streak Virus, Wheat Streak Mosaic Virus and Soil-Borne Wheat Mosaic Virus. Journal of Virological Methods. 148:17-24. Interpretive Summary: The group of Barley and Cereal Yellow Dwarf viruses (YDV) have the potential to significantly reduce the yield and quality of wheat, barley and oat. However, it is not known to what extent the five viruses in this group are responsible for the yield losses. In addition to YDV, there are three other viruses which also are important viral pathogens of wheat. There are some antibody and DNA-based detection methods for these viruses but currently a single test that can detect these important pathogens does not exist. This research developed a method that can simultaneously detect and identify which of these 8 viruses is present in a plant. This study fulfills the need for a rapid and specific small grain cereal virus diagnostic tool that will be extremely useful to plant diagnostic laboratories and Plant Pathologists. This method also has the potential for investigating the epidemiology of these viral diseases in fields growing these cereal crops. This information will be used by plant breeders to determine which viruses are important pathogens within a region and by the grower to identify preventive measures they may need to take to limit the extent of the yield loss caused by these viruses.
Technical Abstract: Barley and Cereal Yellow Dwarf Viruses (B/CYDVs), Wheat Spindle Streak Mosaic (WSSMV), Soil-Borne Wheat Mosaic (SBWMV) Mosaic Virus and Wheat Streak Mosaic Virus (WSMV) constitute the most economically important group of wheat viruses. In this paper, a multiplex reverse transcription polymerase chain reaction (M-RT-PCR) method was developed for the simultaneous detection and discrimination of eight viruses including five strains of B/CYDVs, WSSMV, SBWMV and WSMV. The protocol uses specific primers sets for each virus producing five distinct fragments 295 bp, 175 bp, 237 bp, 400 bp and 365 bp, indicating the presence of all two strains of BYDVs, -PAV, -MAV, CYDV-RPV and two unassigned Luteoviridae BYDV-SGV and -RMV respectively. This system also readily detected WSSMV, SBWMV and WSMV specific amplicons at 154 bp, 219 bp and 193 bp respectively. The amplification specificity of these primers was tested against a range of field samples from different parts of United States. The protocol also utilizes fluorescently tagged primers which can streamline the detection of each virus through fluorescent capillary electrophoresis. This study fulfills the need for a rapid and specific wheat virus diagnostic tool that also has the potential for investigating the epidemiology of these viral diseases.