Development of multiplex RT-PCR assays for rapid detection and subtyping of influenza type A viruses from clinical specimens

Hee Kyoung Chang, Jeung Hyun Park, Min Suk Song, Taek Kyu Oh, Seok Young Kim, Chul Jung Kim, Hyunggee Kim, Moon Hee Sung, Heon Seok Han, Youn Soo Hahn, Young Ki Choi

Research output: Contribution to journalArticlepeer-review

29 Citations (Scopus)

Abstract

We developed multiplex RT-PCR assays that can detect and identify 12 hemagglutinin (H1-H12) and 9 neuraminidase (N1-N9) subtypes that are commonly isolated from avian, swine, and human influenza A viruses. RT-PCR products with unique sizes characteristic of each subtype were amplified by multiplex RT-PCRs, and sequence analysis of each amplicon was demonstrated to be specific for each subtype with 24 reference viruses. The specificity was demonstrated further with DNA or cDNA templates from 7 viruses, 5 bacteria, and 50 influenza A virus - negative specimens. Furthermore, the assays could detect and subtype up to 105 dilution of each of the reference viruses that had an original infectivity titer of 106 EID50/ml. Of 188 virus isolates, the multiplex RT-PCR results agreed completely with individual RT-PCR subtyping results and with results obtained from virus isolations. Furthermore, the multiplex RT-PCR methods efficiently detected mixed infections with at least two different subtypes of influenza viruses in one host. Therefore, these methods could facilitate rapid and accurate subtyping of influenza A viruses directly from field specimens.

Original languageEnglish
Pages (from-to)1164-1169
Number of pages6
JournalJournal of microbiology and biotechnology
Volume18
Issue number6
Publication statusPublished - 2008 Jun

Keywords

  • Clinical specimens
  • Influenza A virus
  • Multiplex RT-PCR
  • Subtyping

ASJC Scopus subject areas

  • Biotechnology
  • Applied Microbiology and Biotechnology

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