Objective: To establish a sensitive and specific multiplex RT-PCR(MRT-PCR) for the simultaneous detection of influenza virus types and subtypes.
Methods: Primers were designed from highly conserved region of the hemagglutinin(HA) gene of influenza H1N1H3N2 and B virus and MRT-PCR was performed. Additional two pairs of primers were designed to determine the N1 and N2 subtypes of neuramidinase NA of influenza H1N1 and H3N2 virus.
Results: The fragments of HA gene of all types of influenza virus were amplified and there was no cross reaction. The sensitivity of detection of influenza H1N1 and H3N2 virus was 0.10 TCID50/50 microliter by the second PCR and that was 0.01 TCID50/50 microliter for influenza B virus. The NA gene of influenza H1N1 and H3N2 virus was also amplified by this method.
Conclusion: The sensitivity of detection of influenza virus from clinical patients' throat washing specimens by MRT-PCR was higher than that by MDCK cell culture or egg embryo isolation. This method was highly sensitive and timely for detection of influenza virus types and subtypes.