A duplex real-time RT-PCR assay for detecting H5N1 avian influenza virus and pandemic H1N1 influenza virus

In March and April 2009, a novel swine-origin pandemic H1N1 influenza virus appeared and spread worldwide. The pandemic of H1N1 2009 influenza virus poses a public health threat. According to the World Health Organization (WHO), as of 30 April 2010, worldwide at least 17919 death cases have been reported [1]. Compared with influenza A H1N1 virus, the highly pathogenic avian influenza virus H5N1 has a much higher mortality rate: H5N1 has a mortality rate of more than 50% while novel pandemic H1N1 has only about 1% [2]. Nevertheless, pandemic H1N1 and highly pathogenic H5N1 manifest similar clinical symptoms at the early stage of infection [3]. Early detection of these two pathogens is an essential prerequisite for effective control and prevention of the pandemic.Real-time RT-PCR/PCR is a powerful method for the sensitive and specific detection of virus-derived nucleic acids in clinical samples [4]. It is time-saving and more specific compared with endpoint PCR. Therefore, real-time RT-PCR assay has been widely used and recommended by WHO for many rapid viral pathogen detection. Moreover, multiplex assays allow to measure several fluorophores in one well, which can simultaneously detect different target sequences [5]. Since the outbreak of pandemic H1N1 influenza, many real-time RT-PCR assays have been developed for detecting of the novel H1N1 [6-13]. Some multiplex real-time RT-PCR assays for simultaneous typing (A/B) and subtyping of H1, H2, H3, H5, H7 and H9 of influenza A viruses have also been reported [14-17]. However, no studies were reported for subtyping of the novel pandemic H1N1 and H5N1 simultaneously.In this study, a duplex TaqMan real-time RT-PCR assay was improved by adjusting the concentrations of primers and probes in the WHO protocols. The assay could simultaneously detect H5N1 avian influenza virus and pandemic H1N1 influenza virus, which could be used for early diagnosis of influenza-like patients and for epidemiological surveillance.The primers an