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Background: Influenza viruses can generate novel reassortants in coinfected cells. The global circulation and occasional introductions of pandemic H1N1/2009 virus in humans and in pigs, respectively, may allow this virus to reassort with other influenza viruses. These possible reassortment events might alter virulence and/or transmissibility of the new reassortants. Investigations to detect such possible reassortants should be included as a part of pandemic influenza surveillance plans., Methods: We established a real-time reverse-transcription (RT)-PCR-based strategy for the detection of reassortment of pandemic H1N1/2009 virus. Singleplex SYBR green-based RT-PCR assays specific for each gene segment of pandemic H1N1/2009 were developed. These assays were evaluated with influenza viruses of various genetic backgrounds., Results: All human pandemic H1N1 (n = 27) and all seasonal human (n = 58) isolates were positive and negative, respectively, for all 8 segments. Of 48 swine influenza viruses isolated from our ongoing surveillance program of influenza viruses in swine, 10 were positive in all reactions. All 8 viral segments of these 10 samples were confirmed to be of pandemic H1N1 origin, indicating that these were caused by zoonotic transmissions from human to pigs. The 38 swine viruses that were nonpandemic H1N1/2009 had 1-6 gene segments positive in the tests. Further characterization of these nonpandemic H1N1/2009 swine viruses indicated that these PCR-positive genes were the precursor genes of the pandemic H1N1/2009 virus., Conclusions: Our results demonstrated that these assays can detect reintroductions of pandemic H1N1/2009 virus in pigs. These assays might be useful screening tools for identifying viral reassortants derived from pandemic H1N1/2009 or its precursors.