Simultaneous Detection of Influenza Viruses A, B, and Swine Origin Influenza A Using Multiplex One-Step Real-Time RT-PCR Assay S. H. R. Monavari & H. R. Mollaie & M. Fazlalipour Received: 16 July 2013 / Accepted: 3 October 2013 # Springer Science+Business Media New York 2013 Abstract Every year, seasonal epidemics of influenza viruses are causing considerable morbidity and mortality worldwide. Also infrequent novel and rearranged strains of influ- enza viruses have caused quick, acute universal pandemics resulting in millions of mortal- ities. The usage of efficient and accurate detection is superior for infection control, effective treatment, and epidemiological supervision. Therefore, evaluation of useful real-time PCR molecular tests for the detection of pandemic viruses is important before the next wave of the pandemic. A novel quantitative real-time reverse-transcription polymerase chain reaction (qRT-PCR) assay with specific primers was used successfully for detection and monitoring of the influenza A, B, and swine influenza. The newly designed primers target highly conserved regions in influenza viruses. Our qRT-PCR assay is highly specific for detecting influenza A, B, and swine influenza viruses. The cutoff CT value was determined <38 for domestic human diagnostic test, under conditions of FDA emergency, and the reaction efficiency of the InfA, swInfA, and InfB assays were thereby estimated to be 97.9 % (R2=0.998), 98.3 % (R2=0.986), and 99.5 % (R2=0.995), respectively. Interestingly, based on our finding, there is no cross reactivity of detecting other viruses. Keywords Influenza A . Influenza B . Swine influenza . rRT-PCR . Real-time PCR Introduction Influenza A and B viruses cause the majority of viral lower respiratory tract infections; elderly and compromised individuals are especially at risk of developing severe illness and complications [1, 2]. Appl Biochem Biotechnol DOI 10.1007/s12010-013-0583-6 S. H. R. Monavari (*) Department of Virology and Anti Microbial Resistance Research Center, Iran University of Medical Sciences, Hemmat Highway, Tehran, Iran e-mail: hrmonavari@yahoo.com H. R. Mollaie : M. Fazlalipour Department of Virology, Iran University of Medical Sciences, Tehran, Iran