One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.
One-step real-time reverse transcription-PCR assays for detecting and subtyping pandemic influenza A/H1N1 2009, seasonal influenza A/H1N1, and seasonal influenza A/H3N2 viruses.
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DOI：
10.1016/j.jviromet.2010.10.018
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发表时间：
2011-01
影响因子：
3.1
通讯作者：
Kageyama T
中科院分区：
文献类型：
--
作者：
Nakauchi M;Yasui Y;Miyoshi T;Minagawa H;Tanaka T;Tashiro M;Kageyama T
2009年甲型H1N1流感大流行（A/H1N1 pdm）病毒在全球范围内造成了重大疫情。针对一步法实时荧光定量PCR（real-time reverse transcription-PCR，rRT-PCR）检测A/H1N1 pdm病毒（H1 pdmrRT-PCR）探针熔解温度低、对病毒变异耐受性差的缺点，对该方法进行了改进。为了帮助筛查A/H1N1 pdm病毒，还开发了用于检测人类季节性A/H1N1（H1 rRT-PCR检测）和A/H3 N2流感病毒（H3 rRT-PCR检测）的rRT-PCR检测。使用体外转录的对照RNA、分离的病毒和其他呼吸道病原性病毒对H1 pdm、H1和H3 rRT-PCR检测进行了评价，结果显示其具有高灵敏度、良好线性（R2 = 0.99）和高特异性。此外，改进的H1 pdm rRT-PCR检测方法可以检测两种A/H1N1 pdm病毒株，即A/Aichi/472/2009（H1N1）pdm和A/Sakai/89/2009（H1N1）pdm，这两种病毒株在血凝素基因的探针结合区发生突变，而不丧失灵敏度。使用开发的三种rRT-PCR检测试剂盒，对2009年5月至10月期间采集的90份临床标本进行了检测。其中，分别有26、20和2份样本被鉴定为A/H1 pdm、A/H3和A/H1阳性，而42份样本为甲型流感病毒阴性。本研究结果表明，这些高灵敏度和特异性的H1 pdm，H1和H3的rRT-PCR检测不仅是有用的诊断流感病毒，但也用于流感病毒的监测。
Pandemic influenza A/H1N1 2009 (A/H1N1pdm) virus has caused significant outbreaks worldwide. A previous one-step real-time reverse transcription-PCR (rRT-PCR) assay for detecting A/H1N1pdm virus (H1pdm rRT-PCR assay) was improved since the former probe had a low melting temperature and low tolerance to viral mutation. To help with the screening of the A/H1N1pdm virus, rRT-PCR assays were also developed for detecting human seasonal A/H1N1 (H1 rRT-PCR assay) and A/H3N2 influenza viruses (H3 rRT-PCR assay). H1pdm, H1, and H3 rRT-PCR assays were evaluated using in vitro-transcribed control RNA, isolated viruses, and other respiratory pathogenic viruses, and were shown to have high sensitivity, good linearity (R2 = 0.99), and high specificity. In addition, the improved H1pdm rRT-PCR assay could detect two viral strains of A/H1N1pdm, namely, A/Aichi/472/2009 (H1N1)pdm and A/Sakai/89/2009 (H1N1)pdm, which have mutation(s) in the probe-binding region of the hemagglutinin gene, without loss of sensitivity. Using the three rRT-PCR assays developed, 90 clinical specimens collected between May and October 2009 were then tested. Of these, 26, 20, and 2 samples were identified as positive for A/H1pdm, A/H3, and A/H1, respectively, while 42 samples were negative for influenza A viruses. The present results suggest that these highly sensitive and specific H1pdm, H1, and H3 rRT-PCR assays are useful not only for diagnosing influenza viruses, but also for the surveillance of influenza viruses.
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