Library of Prefabricated Locked Nucleic Acid Hydrolysis Probes Facilitates Rapid Development of Reverse-Transcription Quantitative Real-Time PCR Assays for Detection of Novel Influenza A/H1N1/09Virus
Library of Prefabricated Locked Nucleic Acid Hydrolysis Probes Facilitates Rapid Development of Reverse-Transcription Quantitative Real-Time PCR Assays for Detection of Novel Influenza A/H1N1/09Virus
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DOI：
10.1373/clinchem.2009.136192
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发表时间：
2009-12-01
影响因子：
9.3
通讯作者：
Reischl, Udo
中科院分区：
文献类型：
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作者：
Wenzel, Juergen J.;Walch, Heiko;Reischl, Udo
背景：新的甲型H1N1/09流感大流行株的出现清楚地表明了对灵活工具的需求，使得快速开发新的诊断方法成为可能。方法：我们设计了一套基于通用探针文库(UPL)的逆转录定量实时聚合酶链式反应(RT-qPCR)分析方法，该文库由165个预先合成的荧光标记的锁定核酸(LNA)水解探针组成，专门用于检测新型甲型流感病毒。结果：在14种血凝素(HA)和神经氨酸酶(NA)基因检测中，有12种方法无需进一步优化即可从稀释的患者样本中检测到病毒核酸。我们用一组样本表征了两种表现最好的检测方法的诊断特异性，这些样本包括不同来源的人和动物流感病毒株，没有显示出交叉反应。这两种引物/探针组合的诊断灵敏度在100-1000个基因组拷贝/毫升之间。结论：面对新型甲型H1N1/09流感疫情的出现，我们在2天内就建立了一套灵敏、特异的RT-qPCR检测方法，用于疑似病例的实验室诊断。H1N1/09作为一种模式，显示了UPL方法用于加速开发新的诊断化验以检测新出现的病原体的可行性。
BACKGROUND: The emergence of a novel pandemic human strain of influenza A (H1N1/09) has clearly demonstrated the need for flexible tools enabling the rapid development of new diagnostic methods.METHODS: We designed a set of reverse-transcription quantitative real-time PCR (RT-qPCR) assays based on the Universal ProbeLibrary (UPL)-a collection of 165 presynthesized, fluorescence-labeled locked nucleic acid (LNA) hydrolysis probes-specifically to detect the novel influenza A virus. We evaluated candidate primer/UPL-probe pairs with 28 novel influenza A/H1N1/09 patient samples of European and Mexican origin.RESULTS: Of 14 assays in the hemagglutinin (HA) and neuraminidase (NA) genes, 12 detected viral nucleic acids from diluted patient samples without need for further optimization. We characterized the diagnostic specificity of the 2 best-performing assays with a set of samples comprising various influenza virus strains of human and animal origin that showed no cross-reactivity. The diagnostic sensitivity of these 2 primer/probe combinations was in the range of 100-1000 genomic copies/mL. In comparison to a reference assay recommended by the German health authorities, the analytical sensitivities and specificities of the assays were equivalent.CONCLUSIONS: Facing the emergence of novel influenza A/H1N1/09, we were able to develop, within 2 days, a set of sensitive and specific RT-qPCR assays for the laboratory diagnosis of suspected cases. H1N1/09 served as a model to show the feasibility of the UPL approach for the expedited development of new diagnostic assays to detect emerging pathogens.