The Simultaneou Detection Of Barley Yellow Dwarf Viruses By Multiplex PCR In Guangzhong Region And Population Distribution

Wheat is one of the most important grain crops worldwide and China ranks first in wheat planting area in the world. However, virus diseases reduce wheat yield and quality; it also leads to serious economic losses. Barley Yellow dwarf virus (BYDV), a group of Luteoviruses and Poleroviruses, constitute one of the most eco wheat grain productions (nomically important viral pathogens of wheat because of their significant negative impact on worldwide. Reaction components and reaction cycling parameters were optimized for establishing a multiplex PCR system to simultaneous detection of barley yellow dwarf viruses, PAV, GPV and GAV. In this paper, a multiplex reverse transcription polymerase chain reaction (M-RT-PCR) method was developed for the simultaneous detection and discrimination of three strains of BYDV. The protocol uses specific primer sets for each virus producing three distinct fragments 274, 342, and 600 bp, indicating the presence of three strains of BYDV, GAV, GPV and PAV. Using three sets of specific primers designed according to the converted sequences of these three viruses respectively, with the reaction of the main ingredients of M-RT-PCR: Primers, Mg2+, Buffer, dNTPs and annealing temperature examined and optimized, expected fragments were successfully amplified by this M- PCR system. The M-PCR provides a simple and rapid method for simultaneous detection of BYDV showing the convenience of this protocol. At the same time, we finished the distribution of BYDV of Guanzhong region in shaan’xi.Virus diseases of wheat often result from mixed infection and thus the M-PCR system that this study developed through many optimizations was able to simultaneously detect virus diseases, so that it could greatly raise the test efficiency and reduce the test cost. In China, there have been no reports about simultaneous detection of BYDV yet. This technique has been successfully employed to test wheat virus diseases resulting from mixed infection.We anticipate that this M-PCR assay will greatly facilitate wheat virus testing for both quarantine and breeding purposes. The increased sensitivity of this molecular assay will help identify wheat breeding lines that are truly resistant to any one, some, or all these viruses. Prior to the development of this assay it was far too costly and time-consuming to determine the distribution of wheat viruses within a field and across geographical regions. Because of the advantages in detecting multiple viruses in a single tube, this assay will be applied toward studying the prevalence and distribution of these viruses leading to a better understanding of the epidemiology of these diseases in China.