Development of a Multiplex RT-PCR Assay for Simultaneous Detection of Ten Major Viral Pathogens of Wheat

Abstract

Triticum mosaic virus (TriMV) and High plains virus (HPV), identified recently, have been considered among the major viruses that affect wheat. Carried by the same vector, wheat curl mite, both of these viruses cause yellowing and stunting of plants which are very similar to many other viruses attacking wheat. This makes it difficult to detect these viruses in different wheat lines, posing a major problem in the yield. This paper highlights the addition of these two viruses to a multiplex RT-PCR based method which already detected the presence of barley and cereal yellow dwarf viruses (B/CYDVs), soil-borne wheat mosaic virus (SBWMV), wheat spindle streak mosaic virus (WSSMV), and wheat streak mosaic virus (WSMV). The method uses specific sets of primers that detect the target viruses TriMV and HPV at 560 bp and 490 bp, respectively, in the presence of other distinct viruses such as B/CYDVs -PAV, -MAV, -SGV, -RPV, -RMV, WSSMV, SBWMV, and WSMV at 295, 175, 237, 400, 365, 154, 219, and 193 bp, respectively. The forward primer for each specific virus was fluorescently tagged to detect it in a higher throughput manner in capillary electrophoresis. All ten viruses may be viewed as peaks in an electropherogram from the capillary electrophoresis corresponding to their product sizes in base pairs. This advancement in the protocol allows detection of all ten wheat viruses in a single test, thus improving the diagnostic capability with only a slight increase in cost.