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J. Microbiol. Biotechnol. 2008; 18(6): 1164-1169

Published online June 28, 2008

Copyright © The Korean Society for Microbiology and Biotechnology.

Development of Multiplex RT-PCR Assays for Rapid Detection and Subtyping of Influenza Type A Viruses from Clinical Specimens

Chang, Hee Kyoung , Jeung Hyun Park , Min-Suk Song , Taek-Kyu Oh , Seok-Young Kim , Chul-Jung Kim 1, Hyunggee Kim 2, Moon-Hee Sung 3, Chang, Hee Kyoung 3, Chang, Hee Kyoung 3 and Young-Ki Choi 3*

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: Influenza A virus, multiplex RT-PCR, subtyping, clinical specimens