Preparation of Armored RNA as a Control for Multiplex Real-Time Reverse Transcription-PCR Detection of Influenza and Severe Acute Respiratory Syndrome Coronavirus

Microbiology Laboratory, Hangzhou Center for Disease Control and Prevention, Hangzhou, Zhejiang, 310006, people' republic of China

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	Abstract

The common respiratory viruses including influenza A, influenza B and new emerging severe acute respiratory syndrome (SARS) viruses may cause similar clinical symptoms. Therefore, differential diagnosis of these virus pathogens is frequently required in single clinical samples. In addition, there is an urgent need for non-infectious and stable RNA standards and controls for multivirus detection. In this study, reverse transcription-PCR targeting the RNA of influenza A, influenza B viruses and SARS coronavirus was performed and the resulting products were spliced into a fragment, which was packaged into armored RNA for use as a non-infectious, quantifiable synthetic substitute. Furthermore, in the present study we developed a multiplex real-time RT-PCR assay in which the armored RNA was used as an external positive control, and the three RNA viruses could be detected simultaneously in a single reaction. The detection limit of the multiplex real-time PCR was 10 copies/µl of armored RNA.