Multiplex reverse transcription-PCR for simultaneous surveillance of influenza A and B viruses

Bin Zhou, Yi Mo Deng, John R. Barnes, October M. Sessions, Tsui Wen Chou, Malania Wilson, Thomas J. Stark, Michelle Volk, Natalie Spirason, Rebecca A. Halpin, Uma Sangumathi Kamaraj, Tao Ding, Timothy B. Stockwell, Mirella Salvatore, Elodie Ghedin, Ian G. Barr, David E. Wentworth

Research output: Contribution to journalArticlepeer-review


Influenza A and B viruses are the causative agents of annual influenza epidemics that can be severe, and influenza A viruses intermittently cause pandemics. Sequence information from influenza virus genomes is instrumental in determining mechanisms underpinning antigenic evolution and antiviral resistance. However, due to sequence diversity and the dynamics of influenza virus evolution, rapid and high-throughput sequencing of influenza viruses remains a challenge. We developed a single-reaction influenza A/B virus (FluA/B) multiplex reverse transcription-PCR (RTPCR) method that amplifies the most critical genomic segments (hemagglutinin [HA], neuraminidase [NA], and matrix [M]) of seasonal influenza A and B viruses for next-generation sequencing, regardless of viral type, subtype, or lineage. Herein, we demonstrate that the strategy is highly sensitive and robust. The strategy was validated on thousands of seasonal influenza A and B virus-positive specimens using multiple next-generation sequencing platforms.

Original languageEnglish (US)
Pages (from-to)3492-3501
Number of pages10
JournalJournal of Clinical Microbiology
Issue number12
StatePublished - Dec 2017


  • Influenza
  • NGS
  • RT-PCR
  • Surveillance

ASJC Scopus subject areas

  • Microbiology (medical)


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