Simultaneous typing and HA/NA subtyping of influenza A and B viruses including the pandemic influenza A/H1N1 2009 by multiplex real-time RT-PCR

J Virol Methods. 2010 Jul;167(1):37-44. doi: 10.1016/j.jviromet.2010.03.007. Epub 2010 Mar 19.


Pandemic influenza A/H1N1 2009 and seasonal influenza viruses are currently co-circulating worldwide. A rapid, sensitive, and specific assay for distinguishing pandemic influenza A/H1N1 2009 from seasonal influenza viruses and for subtyping seasonal influenza A viruses could aid in the surveillance and control of these viral infections. Here, such a multiplex real-time RT-PCR (rRT-PCR) assay for typing influenza A and B viruses and the pandemic influenza A/H1N1 2009 is developed. This assay can also subtype seasonal influenza A viruses simultaneously. The analytical sensitivity is 10-10(4) copies/reaction. The coefficients of variation of inter-assay and intra-assay are 0.04-0.45% and 0.08-0.97%, respectively. The new multiplex rRT-PCR assay is more sensitive in subtyping seasonal influenza viruses than the conventional PCR techniques. Results obtained with this assay for the detection of pandemic influenza A/H1N1 2009 are highly consistent (96.88%) with those achieved using the US CDC's rRT-PCR protocol. A sample identified as "pandemic influenza A/H1N1 2009 positive" by the US CDC's rRT-PCR was reclassified correctly as subtype H3N2 using this assay. Taken together, this new multiplex rRT-PCR protocol could be an important tool for improving diagnosis and management of the pandemic influenza A/H1N1 2009 and seasonal influenza viruses.

Publication types

  • Evaluation Study
  • Research Support, Non-U.S. Gov't

MeSH terms

  • DNA Primers / genetics
  • Hemagglutinin Glycoproteins, Influenza Virus / genetics
  • Humans
  • Influenza A virus / classification*
  • Influenza A virus / isolation & purification*
  • Influenza, Human / diagnosis*
  • Neuraminidase / genetics
  • Reverse Transcriptase Polymerase Chain Reaction / methods*
  • Sensitivity and Specificity
  • Viral Proteins / genetics


  • DNA Primers
  • Hemagglutinin Glycoproteins, Influenza Virus
  • Viral Proteins
  • NA protein, influenza A virus
  • NA protein, influenza B virus
  • Neuraminidase