Molecular Detection of a Novel Human Influenza (H1N1) of Pandemic Potential by Conventional and Real-Time Quantitative RT-PCR Assays
Molecular Detection of a Novel Human Influenza (H1N1) of Pandemic Potential by Conventional and Real-Time Quantitative RT-PCR Assays
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DOI：
10.1373/clinchem.2009.130229
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发表时间：
2009-08-01
影响因子：
9.3
通讯作者：
Peiris, J. S. M.
中科院分区：
文献类型：
--
作者：
Poon, Leo L. M.;Chan, K. H.;Peiris, J. S. M.
背景：甲型流感病毒是医学上重要的病毒病原体，在全世界造成显着的死亡率和发病率。最近出现的一种新型人类甲型流感病毒（H1N1）对健康构成了严重威胁。迫切需要快速检测该病毒的分子检测。方法：我们开发了传统的 I 步 RT-PCR 检测方法和 I 步定量实时 RT-PCR 检测方法来检测新型 H1N1 病毒，但不检测季节性 H1N1 病毒。我们还开发了一种额外的实时 RTPCR，可以将新型 H1N1 与其他猪和人 H I 亚型病毒区分开来。 结果：所有测定的阳性对照检出限均在组织培养中值感染剂量的 1.0 x 10(-4) 至 2.0 x 10(-3) 范围内。检测特异性较高，常规检测和实时检测的所有阴性对照样本均为阴性，包括 7 种人类季节性 H1N1 病毒、1 种人类 H2N2 病毒、2 种人类季节性 H3N2 病毒、1 种人类 H5N1 病毒、7 种禽流感病毒（HA 亚型 4、5、7、8、9 和 10）以及 48 份非流感呼吸道患者的鼻咽抽吸物 (NPA)疾病；对于区分新型 H1N1 与其他猪和人类 H1 亚型病毒的检测，所有阴性对照也均为阴性，包括 20 个对照 NPA、2 个季节性人类 H1N1 病毒、2 个季节性人类 H3N2 病毒和 2 个人类 H5N1 病毒。结论：这些检测似乎有助于快速诊断新型 H1N1 病毒病例，从而更好地做好大流行准备。
BACKGROUND: Influenza A viruses are medically important viral pathogens that cause significant mortality and morbidity throughout the world. The recent emergence of a novel human influenza A virus (H1N1) poses a serious health threat. Molecular tests for rapid detection of this virus are urgently needed.METHODS: We developed a conventional I-step RT-PCR assay and a I-step quantitative real-time RT-PCR assay to detect the novel H1N1 virus, but not the seasonal H1N1 viruses. We also developed an additional real-time RTPCR that can discriminate the novel H1N1 from other swine and human H I subtype viruses.RESULTS: All of the assays had detection limits for the positive control in the range of 1.0 x 10(-4) to 2.0 x 10(-3) of the median tissue Culture infective dose. Assay specificities were high, and for the conventional and real-time assays, all negative control samples were negative, including 7 human seasonal H1N1 viruses, I human H2N2 virus, 2 human seasonal H3N2 viruses, 1 human H5N1 virus, 7 avian influenza viruses (HA subtypes 4,5,7, 8,9,and 10), and 48 nasopharyngeal aspirates (NPAs) from patients with noninfluenza respiratory diseases; for the assay that discriminates the novel H1N1 from other swine and human H1 subtype viruses, all negative controls were also negative, including 20 control NPAs, 2 seasonal human H1N1 viruses, 2 seasonal human H3N2 viruses, and 2 human H5N1 viruses.CONCLUSIONS: These assays appear useful for the rapid diagnosis of cases with the novel H1N1 virus, thereby allowing better pandemic preparedness.