Cloning of HA, NA and M gene of H5N1 avian influenza virus and preparation of RNA fragments with transcription in vitro

Objective To transcript RNA in vitro in order to obtain quantitative standard for detecting mRNA of H5N1 avian influenza virus.Methods According to the specific sequence of HA/NA/M gene of H5N1 avian influenza virus,the primers were designed and synthesized.The fragments generated by RT-PCR were cloned into the pGEM-T Easy vector with T7 cloning protocol.The positive recombinant linearized plasmids were used to transcript RNA in vitro and the RNA was used as standard quantitative template of RT-PCR method.Results Specific framents were amplified from virus culture medium,which were confirmed by DNA cloning and sequencing.The fulllength cRNA of HA/NA/M gene of H5N1 avian influenza virus with precise quantification copy number was obtained successfully.Conclusion The HA/NA/McRNA obtained could be used as quantitative standard for rapid detection of nucleic acid of H5N1 avian influenza virus.