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KMID : 0545120080180061164
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Journal of Microbiology and Biotechnology
2008 Volume.18 No. 6 p.1164 ~ p.1169
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Development of Multiplex RT-PCR Assays for Rapid Detection and Subtyping of Influenza Type A Viruses from Clinical Specimens
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Chang Hee-Kyoung
Park Jeung-Hyun
Song Min-Suk
Oh Taek-Kyu
Kim Chul-Joong
Kim Hyung-Gee
Sung Moon-Hee
Han Heon-Seok
Hahn Youn-Soo
Choi Young-Ki
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Abstract
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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 virus뻨egative 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.
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KEYWORD
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Influenza A virus, multiplex RT-PCR, subtyping, clinical specimens
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