Rapid Discrimination of Oseltamivir-Resistant 275Y and -Susceptible 275H Substitutions in the Neuraminidase Gene of Pandemic Influenza A/H1N1 2009 Virus by Duplex One-Step RT-PCR Assay
Rapid Discrimination of Oseltamivir-Resistant 275Y and -Susceptible 275H Substitutions in the Neuraminidase Gene of Pandemic Influenza A/H1N1 2009 Virus by Duplex One-Step RT-PCR Assay
复制标题
DOI：
10.1002/jmv.22101
复制
发表时间：
2011-07-01
影响因子：
12.7
通讯作者：
Kageyama, Tsutomu
中科院分区：
文献类型：
--
作者：
Nakauchi, Mina;Ujike, Makoto;Kageyama, Tsutomu
2009年甲型H1N1流感大流行（A/H1N1 pdm）病毒去年（2009年）在全球范围内造成了重大疫情。包括日本在内的几个国家零星报告了一些对奥司他韦具有耐药性的A/H1N1 pdm病毒，这些病毒在神经氨酸酶（NA）蛋白中具有H275 Y取代，但它们对扎那米韦敏感，没有在社区中传播。为了快速、简便地检测具有H275 Y基因的奥司他韦耐药A/H1N1 pdm病毒，在终点基因分型分析方法的基础上，建立了双重一步法RT-PCR检测方法（H275 Y RT-PCR检测方法）。H275 Y RT-PCR检测使用几种亚型/类型的甲型和B型流感病毒以及其他呼吸道病原性病毒进行评价，显示具有高灵敏度和高特异性。使用H275 Y RT-PCR检测试剂盒进行RNA纯化后检测了44份临床标本，结果发现1份临床标本含有H275 Y突变病毒。73株临床分离株不经RNA纯化，直接用细胞培养上清中的临床分离株进行H275 Y检测，结果与NA测序结果一致。由于H275 Y RT-PCR检测方法可以在未经RNA纯化的情况下检测临床分离株中的H275 Y突变，以及在RNA纯化后检测临床标本中的H275 Y突变病毒，因此该检测方法被认为是监测筛选奥司他韦耐药A/H1N1 pdm病毒活性的有力工具。J. Med. Virol. 83：1121-1127，2011. (C)2011 Wiley-Liss，Inc.
Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus caused significant outbreaks worldwide last year (2009). A number of oseltamivir-resistant A/H1N1pdm viruses possessing an H275Y substitution in the neuraminidase (NA) protein were reported sporadically in several countries, including Japan, but they were sensitive to zanamivir and did not spread in the community. In this study, to monitor rapidly and simply oseltamivir-resistant A/H1N1pdm viruses possessing H275Y, a duplex one-step RT-PCR assay (H275Y RT-PCR assay) was developed based on an endpoint genotyping analysis method. H275Y RT-PCR assay evaluated using several subtypes/types of influenza A and B viruses and other respiratory pathogenic viruses and shown to have high sensitivity and high specificity. Forty-four clinical specimens were tested after RNA purification using the H275Y RT-PCR assay, resulting in one clinical specimen being found to contain a virus possessing the H275Y mutation. Seventy-three clinical isolates were then tested with the H275Y assay by using clinical isolates in the cultured supernatants of cells directly, without RNA purification, and the results were consistent with the NA sequencing. Since the H275Y RT-PCR assay could detect the H275Y mutation in clinical isolates without RNA purification, as well as a H275Y mutated virus in clinical specimens after RNA purification, the assay was considered a powerful tool for surveillance screening of oseltamivir-resistant A/H1N1pdm virus activity. J. Med. Virol. 83:1121-1127, 2011. (C) 2011 Wiley-Liss, Inc.