Duplex Real-Time Reverse Transcriptase PCR Assays for Rapid Detection and Identification of Pandemic (H1N1) 2009 and Seasonal Influenza A/H1, A/H3, and B Viruses
Duplex Real-Time Reverse Transcriptase PCR Assays for Rapid Detection and Identification of Pandemic (H1N1) 2009 and Seasonal Influenza A/H1, A/H3, and B Viruses
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DOI：
10.1128/jcm.01435-09
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发表时间：
2010-03-01
影响因子：
9.4
通讯作者：
Smith, David W.
中科院分区：
文献类型：
--
作者：
Chidlow, Glenys;Harnett, Gerald;Smith, David W.
2009年4月，美国和墨西哥出现了一种新型甲型H1N1流感病毒的报告，该病毒现已被世界卫生组织指定为2009年H1N1大流行。澳大利亚对大流行的管理需要对大量新型流感毒株标本进行快速和可靠的检测，并与季节性流感毒株进行区分。设计了一种用于检测2009年H1N1流感大流行的实时逆转录酶PCR （RT-PCR）方法，并将其与现有的季节性流感病毒A和b的实时RT-PCR方法一起使用。三种双相RT-PCR检测方法，每一种含有两对引物和相应的5’核酸酶探针，在对照材料和储存样品上进行初步评估，显示出较高的灵敏度和特异性。随后，对11,000多个临床样本进行了甲型和乙型流感基质基因靶点和季节性流感A/H1、A/H3和2009年甲型H1N1流感大流行的特异性血凝素基因靶点检测。2009年甲型H1N1流感的最低敏感性和特异性分别为98.8%和100%，季节性甲型H1N1流感的最低敏感性和特异性分别为81.5%和98.9%，甲型H1N1流感的最低敏感性和特异性分别为96.3%和99.6%。自动样品提取促进了样品的快速处理，使分析能够准确，快速和具有成本效益的筛选大量临床样品。
Reports of a novel influenza virus type A (H1N1), now designated by the World Health Organization as pandemic (H1N1) 2009, emerged from the United States and Mexico in April 2009. The management of the pandemic in Australia required rapid and reliable testing of large numbers of specimens for the novel influenza strain and differentiation from seasonal influenza strains. A real-time reverse transcriptase PCR (RT-PCR) assay for the detection of pandemic (H1N1) 2009 was designed and used with existing real-time RT-PCR assays for seasonal influenza viruses A and B. MS2 coliphage was added to all samples and amplified as a quality control. Three duplex RT-PCR assays, each containing two primer pairs and corresponding 5' nuclease probes, were initially evaluated on control material and stored samples and showed high sensitivity and specificity. More than 11,000 clinical samples were then tested for influenza A and B matrix gene targets and specific hemagglutinin gene targets for seasonal influenza A/H1, A/H3, and pandemic A (H1N1) 2009. Minimum sensitivities and specificities were 98.8% and 100%, respectively, for pandemic (H1N1) 2009, 81.5% and 98.9% for seasonal A/H1, and 96.3% and 99.6% for A/H3. Automated sample extraction facilitated the rapid processing of samples so that the assays allowed accurate, rapid, and cost-effective screening of large numbers of clinical samples.