5. Conclusion
In this study, we first tried to design four multiplex primers with four possible combinations of eight segments to detect EIV using a multiplex RT-PCR. The candidate primers suggested in this study have been proved to be highly specific to the target lineage with satisfactory analytical results in silico and in vitro. Even though the current study was limited to detect equine influenza virus, it is expected that the strategies suggested to design and test primers in this study could be further applied for the detection of other influenza A viruses from different hosts. Improving the PCR conditions and making more various combinations with two or three primer pairs could be possible depending on the purpose of the studies. We expect that the approach could have a wide range of applications in monitoring and diagnosing newly emerging EIV. Finally, attempts to test multiplex RT-PCR with more recent viral samples as well as periodic evaluation to detect any changes in the primer binding sites are under way.
. 3).