Rapid Detection of Avian Influenza virus H5 by Real-Time RT-PCR and TaqMan-MGB probe

A real-time reverse transcription-polymerase chain reaction (RRT-PCR) assay based on TaqMan-MGB probe was developed to rapidly detect avain influenza virus subtype H5. The assay, which was based on primers and TaqMan-MGB probes selected from highly conserved regions of the hemagglutinin gene of avain influenza virus subtype H5, was optimized in a reaction system and a condition to improve the sensitivity, specificity and accuracy. In addition, colone technology was used to develop a quantitative PCR format with virus copies amount. The results showed that the best concentration of primers and probe was 640nmol/L and 480nmol/L, respectively. None of the negative control samples showed false-positive reactions when done in duplicates. The detection limit of the assay was 100 copies per reaction. A linear standard curve was obtained between 102 and 107 DNA copies/reaction. It took only three hours from viral RNA extraction to complete the detection. The assay was simple and highly reproducible. This real-time RT-PCR assay is an excellent method suitable for rapid and quantitative detection of avian influenza virus H5 under clinical conditions.