Journal of Microbiology and Biotechnology

The Korean Society for Microbiology and Biotechnology (한국미생물·생명공학회)
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Development of Multiplex RT-PCR Assays for Rapid Detection and Subtyping of Influenza Type A Viruses from Clinical Specimens

  • Chang, Hee-Kyoung (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Park, Jeung-Hyun (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Song, Min-Suk (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Oh, Taek-Kyu (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Seok-Young (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Kim, Chul-Jung (College of Veterinary Medicine, Chungnam National University) ;
  • Kim, Hyung-Gee (Division of Bioscience and Technology, College of Life and Environmental Science, Korea University) ;
  • Sung, Moon-Hee (Department of Bio and Nano Chemistry, College of Natural Sciences, Kookmin University) ;
  • Han, Heon-Seok (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Hahn, Youn-Soo (College of Medicine and Medical Research Institute, Chungbuk National University) ;
  • Choi, Young-Ki (College of Medicine and Medical Research Institute, Chungbuk National University)
  • Published : 2008.06.30
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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 $10^5$ dilution of each of the reference viruses that had an original infectivity titer of $10^6\;EID_{50}/ml$. 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.

Keywords

References

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