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Influenza A virus subtype H5N1 causes a rapidly fatal systemic disease in domestic poultry and spreads directly from poultry to humans. The aim of this study was to develop a rapid, cost-saving and effective method for influenza A virus subtype H5N1 detection. The selected primer set was used in single-step RT-PCR for simultaneous detection in multiplex format of the 276-, 189-, and 131-bp fragments, corresponding to sequences specific for M, H5 and N1. The amplified DNA fragments were clearly separated by agarose gel electrophoresis. The sensitivity of this assay was about 10³copies/µL. Moreover, this method can be applied to detect not only avian but also human influenza A virus subtype H5N1. In conclusion, the highlights of this particular method are its rapidity and cost-effectiveness, thus rendering it feasible and attractive for large-scale screening at times of influenza A virus subtype H5N1 outbreak.