Nanopore sequencing reveals novel targets for detection and surveillance of human and avian influenza a viruses

Cyril Chik Yan Yip, Wan Mui Chan, Jonathan Daniel Ip, Claudia Win May Seng, Kit Hang Leung, Rosana Wing Shan Poon, Anthony Chin Ki Ng, Wai Lan Wu, Hanjun Zhao, Kwok Hung Chan, Gilman Kit Hang Siu, Timothy Ting Leung Ng, Vincent Chi Chung Cheng, Kin Hang Kok, Kwok Yung Yuen, Kelvin Kai Wang To

Research output: Journal article publicationJournal articleAcademic researchpeer-review

21 Citations (Scopus)


Accurate detection of influenza A virus (IAV) is crucial for patient management, infection control, and epidemiological surveillance. The World Health Organization and the Centers for Disease Control and Prevention have recommended using the M gene as the diagnostic gene target for reverse-transcription-PCR (RT-PCR). However, M gene RT-PCR has reduced sensitivity for recent IAV due to novel gene mutations. Here, we sought to identify novel diagnostic targets for the molecular detection of IAV using long-read third-generation sequencing. Direct nanopore sequencing from 18 nasopharyngeal specimens and one saliva specimen showed that the 5= and 3= ends of the PB2 gene and the entire NS gene were highly abundant. Primers selected for PB2 and NS genes were well matched with seasonal or avian IAV gene sequences. Our novel PB2 and NS gene real-time RT-PCR assays showed limits of detection similar to or lower than that of M gene RT-PCR and achieved 100% sensitivity and specificity in the detection of A(H1N1), A(H3N2), and A(H7N9) in nasopharyngeal and saliva specimens. For 10 patients with IAV detected by M gene RT-PCR conversion in sequentially collected specimens, NS and/or PB2 gene RT-PCR was positive in 2 (20%) of the initial specimens that were missed by M gene RT-PCR. In conclusion, we have shown that PB2 or NS gene RT-PCRs are suitable alternatives to the recommended M gene RT-PCR for diagnosis of IAV. Long-read nanopore sequencing facilitates the identification of novel diagnostic targets.

Original languageEnglish
Article numbere02127-19
JournalJournal of Clinical Microbiology
Issue number5
Publication statusPublished - 23 Apr 2020


  • Diagnostic assay
  • Influenza
  • Next-generation sequencing

ASJC Scopus subject areas

  • Microbiology (medical)


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