Chang Hee Kyoung, Park Jeung Hyun, Song Min-Suk, Oh Taek-Kyu, Kim Seok-Young, Kim Chul-Jung, Kim Hyunggee, Sung Moon-Hee, Han Heon-Seok, Hahn Youn-Soo, Choi Young-Ki
College of Medicine and Medical Research Institute, Chungbuk National University, Cheongju, Korea.
J Microbiol Biotechnol. 2008 Jun;18(6):1164-9.
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.
我们开发了多重逆转录聚合酶链反应（RT-PCR）检测方法，该方法能够检测和鉴定从禽流感、猪流感和人流感 A 病毒中常见分离出的 12 种血凝素（H1-H12）亚型和 9 种神经氨酸酶（N1-N9）亚型。通过多重 RT-PCR 扩增出具有各亚型独特大小特征的 RT-PCR 产物，并且对 24 种参考病毒的每个扩增子进行序列分析，结果表明对每种亚型具有特异性。利用来自 7 种病毒、5 种细菌和 50 份甲型流感病毒阴性标本的 DNA 或 cDNA 模板进一步证明了其特异性。此外，这些检测方法能够检测和分型原始感染滴度为 106 EID50/ml 的每种参考病毒高达 105 倍稀释度的样本。在 188 株病毒分离株中，多重 RT-PCR 结果与单独的 RT-PCR 亚型分型结果以及病毒分离结果完全一致。此外，多重 RT-PCR 方法能够有效检测同一宿主中至少两种不同亚型流感病毒的混合感染。因此，这些方法有助于直接从现场标本中快速、准确地对甲型流感病毒进行亚型分型。
